Factor viii polypeptide formulations

ABSTRACT

The present invention provides a formulation of a Factor VIII polypeptide, e.g., FVIII-Fc, and methods of using the same. The FVIII polypeptide can be a recombinant FVIII protein, a short-acting FVIII protein, or a long-acting FVIII protein. The pharmaceutical formulation comprising a FVIII polypeptide can be used for individual prophylaxis, weekly prophylaxis, episodic (on-demand) treatment, or perioperative management of hemophilia.

REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 15/455,043, filed Mar. 9, 2017, which is a division of U.S.patent application Ser. No. 14/213,180, filed Mar. 14, 2014, now U.S.Pat. No. 9,623,088, which claims the benefit of U.S. Provisional PatentApplication Ser. Nos. 61/800,293, filed Mar. 15, 2013, 61/817,085, filedApr. 29, 2013, 61/829,884, filed May 31, 2013, 61/839,477, filed Jun.26, 2013, 61/863,860, filed Aug. 8, 2013, 61/876,927, filed Sep. 12,2013, 61/879,955, filed Sep. 19, 2013, and 61/897,742, filed Oct. 30,2013, all of which are incorporated herein by reference in theirentireties.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The content of the electronically submitted sequence listing in ASCIItext file (Name: 709032_SA9-436USDIVCON_ST25.txt; Size: 61,753 bytes;and Date of Creation: Aug. 14, 2020) is incorporated herein by referencein its entirety.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates generally to the field of therapeutics forhemostatic disorders.

Background Art

Hemophilia A is an X-linked bleeding disorder caused by mutations and/ordeletions in the factor VIII (FVIII) gene resulting in a deficiency ofFVIII activity (Peyvandi, F. el al. Haemophilia 12:82-89 (2006). Thedisease is characterized by spontaneous hemorrhage and excessivebleeding after trauma. Over time, the repeated bleeding into muscles andjoints, which often begins in early childhood, results in hemophilicarthropathy and irreversible joint damage. This damage is progressiveand can lead to severely limited mobility of joints, muscle atrophy andchronic pain (Rodriguez-Merchan, E. C., Semin. Thromb. Hemost. 29:87-96(2003), which is herein incorporated by reference in its entirety).

The A2 domain is necessary for the procoagulant activity of the factorVIII molecule. Studies show that porcine factor VIII has six-foldgreater procoagulant activity than human factor VII (Lollar, P., and E.T. Parker, J. Biol. Chem. 266:12481-12486 (1991)), and that thedifference in coagulant activity between human and porcine factor VIIIappears to be based on a difference in amino acid sequence between oneor more residues in the human and porcine A2 domains (Lollar, P., etal., J. Biol. Chem. 26:23652-23657 (1992)), incorporated herein byreference in its entirety.

Treatment of hemophilia A is by replacement therapy targetingrestoration of FVIII activity to 1 to 5% of normal levels to preventspontaneous bleeding (Mannucci, P. M., et al., N. Engl. J. Med344:1773-1779 (2001), which is herein incorporated by reference in itsentirety). There are plasma-derived and recombinant FVII productsavailable to treat bleeding episodes on-demand or to prevent bleedingepisodes from occurring by treating prophylactically.

Reduced mortality, prevention of joint damage, and improved quality oflife have been important achievements due to the development ofplasma-derived and recombinant FVIII. Prolonged protection from bleedingwould represent another key advancement in the treatment of hemophilia Apatients.

BRIEF SUMMARY OF THE INVENTION

The present invention comprises a pharmaceutical composition comprising:(a) a FVIII polypeptide; (b) one or more stabilizing agents selectedfrom sucrose, trehalose, raffinose, arginine, or mixture thereof; (c)sodium chloride (NaCl); (d) L-histidine: (e) calcium chloride; and (f)polysorbate 20 or polysorbate 80. In one embodiment, mannitol, glycine,alanine, or hydroxyethyl starch is not included in the pharmaceuticalcomposition. In another embodiment, NaCl is the only bulking agent. Inother embodiments, the FVII polypeptide is a long-acting polypeptide ora short-acting polypeptide.

In other embodiments, the pharmaceutical composition comprises about 1%(w/v) to about 2.5% (w/v) sucrose, about 1.3% (w/v) sucrose to about2.0% (w/v) sucrose, about 1.33% (w/v) sucrose or about 2.0% (w/v)sucrose, about 10 mg/m to about 25 mg/m sucrose, about 13 mg/ml to about20 mg/ml sucrose, about 13.3 mg/ml sucrose, or about 20.0 mg/ml sucrose.In still other embodiments, the pharmaceutical composition comprisesabout 150 mM to about 250 mM NaCl, about 175 mM to about 225 mM NaCl,about 200 mM to about 210 mM NaCl, about 205 mM NaCl, about 8.8 mg/ml toabout 14.6 mg/ml NaCl, about 10 mg/ml to about 13 mg/ml NaCl, about 12.0mg/ml NaCl, about 250 mM to about 350 mM NaCl, about 275 mM to about 325mM NaCl, about 308 mM NaCl, about 14.6 mg/ml to about 20.5 mg/ml NaCl,about 16 mg/mi to about 19 mg/ml NaCl, or about 18.0 mg/ml NaCl.

In yet other embodiments, the pharmaceutical composition comprises about5 mM to about 15 mM L-histidine, about 6.64 mM L-histidine to about 9.8mM L-histidine, about 0.75 mg/ml to about 2.25 mg/ml L-histidine, orabout 1.03 mg/ml L-histidine to about 1.55 mg/ml L-histidine. In certainembodiments, the pharmaceutical composition comprises about 5 mM toabout 10 mM calcium chloride, about 5.4 mM calcium chloride to about 8mM calcium chloride, about 0.75 mg/ml to about 1.5 mg/ml calciumchloride dihydrate, or about 0.8 mg/ml calcium chloride dihydrate toabout 1.18 mg/ml calcium chloride dihydrate.

In some embodiments, the pharmaceutical composition comprises about0.008% (w/v) to about 0.025% (w/v) polysorbate 20 or polysorbate 80,about 0.013% (w/v) polysorbate 20 or polysorbate 80 or about 0.02% (w/v)polysorbate 20 or polysorbate 80, about 0.08 mg/ml to about 0.25 mg/mlpolysorbate 20 or polysorbate 80, about 0.13% mg/ml polysorbate 20 orpolysorbate 80 or about 0.20 mg/ml polysorbate 20 or polysorbate 80.

In other embodiments, the pharmaceutical composition comprises therFVIIIFc polypeptide, which comprises a first subunit comprising anamino acid sequence at least 90% or 95% identical to amino acids 20 to1684 of SEQ ID NO:2 or 20 to 2578 of SEQ ID NO:6 and a second subunitcomprising an amino acid sequence at least 90% to 95% identical to aminoacids 21 to 247 of SEQ ID NO:4. In certain embodiments, wherein therFVIIIFc polypeptide comprises a first subunit comprising amino acids 20to 1684 of SEQ ID NO:2 or 20 to 2578 of SEQ ID NO:6, and a secondsubunit comprising amino acids 21 to 247 of SEQ ID NO:4.

In certain embodiments, a FVIII polypeptide is present in apharmaceutical composition at a concentration of about 50 IU/ml to about2500 IU/ml, e.g., 83 IU/ml, 167 FU/ml, 250 IU/ml, 333 IU/ml, 500 IU/ml,667 IU/ml, 1000 IU/ml, 1333 IU/ml, 1667 IU/ml, or 2000 IU/ml of theFVIII polypeptide. In some embodiments, a FVIII polypeptide is presentin a pharmaceutical composition at a concentration of about 100 IU/ml toabout 4000 IU/ml, e.g., 150 IU/ml, 287.5 IU/ml, 431.25 IU/ml, 575 IU/ml,862.5 IU/ml, 1150 IU/ml, 1725 IU/ml, 2300 IU/ml, 2875 IU/ml, or 3450IU/ml of the FVIII polypeptide.

In other embodiments, a pharmaceutical composition comprises: (a) about50 IU/ml to about 2500 IU/ml of a long-acting FVIII polypeptide; (b)about 1% (w/v) to about 2.5% (w/v) of sucrose; (c) about 150 mM to about250 mM NaCl; (d) about 5 mM to about 15 mM L-histidine; (e) about 5 mMto about 10 mM calcium chloride; and (f) about 0.008% (w/v) to about0.025% of polysorbate 20 or polysorbate 80. In other embodiments, thepharmaceutical composition comprises about 175 mM to about 225 mM NaClor about 200 mM to about 210 mM NaCl.

In yet other embodiments, a pharmaceutical composition comprises (a)about 83 IU/ml, about 167 IU/ml, about 250 IU/ml, about 333 IU/ml, about500 IU/ml, about 667 IU/ml, about 1000 IU/ml, about 1333 IU/ml, about1667 IU/ml, or about 2000 IU/ml of a FVIII polypeptide; (b) about 1.33%(w/v) of sucrose; (c) about 205 mM NaCl; (d) about 6.64 mM L-histidine;(e) about 5.4 mM calcium chloride; and (f) about 0.013% (w/v) ofpolysorbate 20 or polysorbate 80.

In some embodiments, a pharmaceutical composition comprises: (a) about100 IU/ml to about 4000 IU/ml of a FVIII polypeptide; (b) about 1% (w/v)to about 2.5% (w/v) of sucrose; (c) about 250 mM to about 350 mM NaCl;(d) about 5 mM to about 15 mM L-histidine; (e) about 5 mM to about 10 mMcalcium chloride; and (f) about 0.008% (w/v) to about 0.025% ofpolysorbate 20 or polysorbate 80. In other embodiments, thepharmaceutical composition comprises about 275 mM to about 325 mM NaCl.

In certain embodiments, a pharmaceutical composition comprises: (a)about 150 IU/ml, about 287.5 IU/ml, about 431.25 FU/ml, about 575 FU/ml,about 862.5 IU/ml, about 1150 IU/ml, about 1725 IU/ml, about 2300 IU/ml,about 2875 U/ml, or about 3450 IU/ml of a FVIII polypeptide; (b) about2.0% (w/v) of sucrose; (c) about 308 mM NaCl; (d) about 9.8 mML-histidine; (e) about 8 mM calcium chloride; and (f) about 0.020% (w/v)of polysorbate 20 or polysorbate 80. In other embodiments, apharmaceutical composition comprises: (a) about 50 U/mi to about 2500IU/ml of a FVIII polypeptide; (b) about 10 mg/ml to about 25 mg/ml ofsucrose; (c) about 8.8 mg/ml to about 14.6 mg/ml NaCl; (d) about 0.75mg/ml to about 2.25 mg/ml L-histidine; (e) about 0.75 mg/ml to about 1.5mg/ml calcium chloride dihydrate; and (f) about 0.08 mg/ml to about 0.25mg/ml of polysorbate 20 or polysorbate 80. In some embodiments, apharmaceutical composition comprises: (a) about 83 IU/ml, about 167IU/ml, about 250 IU/ml, about 333 IU/ml, about 500 IU/ml, about 667IU/ml, about 1000 IU/ml, about 1333 IU/ml, about 1667 IU/ml, or about2000 IU/ml of a FVIII polypeptide; (b) about 13.3 mg/mi of sucrose; (c)about 12.0 mg/ml NaCl; (d) about 1.03 mg/ml L-histidine; (e) about 0.8mg/ml calcium chloride dihydrate; and (f) about 0.13 mg/ml ofpolysorbate 20 or polysorbate 80.

In other embodiments, a pharmaceutical composition comprises: (a) about100 IU/ml to about 4000 IU/ml of a FVIII polypeptide; (b) about 10 mg/mlto about 25 mg/ml of sucrose; (c) about 14.6 mg/ml to about 20.5 mg/mlNaCl; (d) about 0.75 mg/ml to about 2.25 mg/ml L-histidine; (e) about0.75 mg/ml to about 1.5 mg/ml calcium chloride dihydrate; and (f) about0.08 mg/ml to about 0.25 mg/ml of polysorbate 20 or polysorbate 80. Incertain embodiments, a pharmaceutical composition comprises: (a) about150 IU/ml, about 287.5 IU/ml, about 431.25 IU/ml, about 575 IU/ml, about862.5 IU/ml, about 1150 IU/ml, about 1725 IU/ml, about 2300 IU/ml, about2875 IU/ml, or about 3450 IU/ml of a FVIII polypeptide; (b) about 20.0mg/ml of sucrose; (c) about 18.0 mg/ml NaCl; (d) about 1.55 mg/mlL-histidine; (e) about 1.18 mg/ml calcium chloride dihydrate; and (f)about 0.20 mg/ml of polysorbate 20 or polysorbate 80.

In some embodiments, the present invention includes a pharmaceutical kitcomprising: (a) a first container comprising a lyophilized powder,wherein the powder comprises (i) a FVIII polypeptide, (ii) one or morestabilizing agents selected from sucrose, trehalose, raffinose,arginine, or mixture thereof; (iii) sodium chloride (NaCl); (iv)L-histidine; (v) calcium chloride; and (vi) polysorbate 20 orpolysorbate 80; and (b) a second container comprising sterilized waterfor injections to be combined with the lyophilized powder of the firstcontainer. In other embodiments, a pharmaceutical kit comprises: (a) afirst container comprising a lyophilized powder, where the powdercomprises (i) about 250 IU, about 500 IU, about 750 IU, about 1000 IU,about 1500 IU, about 2000 IU, about 3000 IU, about 4000 IU, about 5000IU, or about 6000 IU of a FVIII polypeptide, (ii) about 40 mg ofsucrose; (iii) about 36 mg of sodium chloride; (iv) about 3.1 mg ofL-histidine; (v) about 2.40 mg of calcium chloride dihydrate; and (v)about 0.40 mg of polysorbate 20 or polysorbate 80; and (b) a secondcontainer comprising sterilized water for injections at a volumesufficient to produce, when combined with the lyophilized powder of thefirst container, a solution comprising: (i) about 83 IU/ml, about 167IU/ml, about 250 IU/ml, about 333 IU/ml, about 500 IU/ml, about 667IU/ml, about 1000 IU/ml, about 1333 IU/ml, about 1667 IU/ml, or about2000 IU/ml of a FVIII polypeptide, respectively; (ii) about 1.33% (w/v)of sucrose; (iii) about 205 mM NaCl; (iv) about 6.64 mM L-histidine; (v)about 5.4 mM of calcium chloride; and (vi) about 0.013% (w/v) ofpolysorbate 20 or polysorbate 80.

In other embodiments, a pharmaceutical kit comprises: (a) a firstcontainer comprising a lyophilized powder, where the powder comprises(i) about 250 IU, about 500 IU, about 750 IU, about 1000 IU, about 1500IU, about 2000 IU, about 3000 IU, about 4000 IU, about 5000 IU, or about6000 IU of a FVIII polypeptide, (ii) about 40 mg of sucrose; (iii) about36 mg of sodium chloride; (iv) about 3.1 mg of L-histidine; (v) about2.40 mg of calcium chloride dihydrate; and (v) about 0.40 mg ofpolysorbate 20 or polysorbate 80; and (b) a second container comprisingsterilized water for injections at a volume sufficient to produce, whencombined with the lyophilized powder of the first container, a solutioncomprising: (i) about 83 IU/ml, about 167 IU/ml, about 250 IU/ml, about333 IU/ml, about 500 IU/ml, about 667 IU/ml, about 1000 IU/ml, about1333 IU/ml, about 1667 IU/ml, or about 2000 IU/ml of a FVIIIpolypeptide, respectively; (ii) about 13.3 mg/ml of sucrose; (iii) about12.0 mg/ml of NaC; (iv) about 1.03 mg/ml of L-histidine; (v) about 0.80mg/ml of calcium chloride; and (vi) about 0.13 mg/ml of polysorbate 20or polysorbate 80.

The invention also includes a method of reducing or decreasing ableeding episode or treating or preventing a bleeding conditioncomprising administering a pharmaceutical composition comprising a FVIIIpolypeptide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows details on the design of the sequential PK subgroup (Arm 1)dosing and PK sampling.

FIG. 2 shows the mean observed FVIII activity with rFVIIIFc and rFVIIIover time: one stage clotting assay (logarithmic scale) in thesequential pharmacokinetic subgroup. rFVIIIFc demonstrated anapproximate 50% longer elimination half-life and mean residence timecompared with wild-type Factor VIII (a short-acting FVIII polypeptide)(P<0.001).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a formulation for a Factor VIIIpolypeptide, e.g., FVIII-Fc, and methods of using thereof.

1. Definitions

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a”, “an” and “the” include plural referentsunless the context clearly dictates otherwise. The terms “a” (or “an”),as well as the terms “one or more,” and “at least one” can be usedinterchangeably herein.

The term “about” is used herein to mean approximately, roughly, around,or in the regions of. When the term “about” is used in conjunction witha numerical range, it modifies that range by extending the boundariesabove and below the numerical values set forth. In general, the term“about” is used herein to modify a numerical value above and below thestated value by a variance of 10 percent, up or down (higher or lower).

Furthermore, “and/or” where used herein is to be taken as specificdisclosure of each of the two specified features or components with orwithout the other. Thus, the term “and/or” as used in a phrase such as“A and/or B” herein is intended to include “A and B,” “A or B,” “A”(alone), and “B” (alone). Likewise, the term “and/or” as used in aphrase such as “A, B, and/or C” is intended to encompass each of thefollowing embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C;A and C; A and B; B and C; A (alone); B (alone); and C (alone).

It is understood that wherever embodiments are described herein with thelanguage “comprising,” otherwise analogous embodiments described interms of “consisting of” and/or “consisting essentially of” are alsoprovided.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure is related. For example, the ConciseDictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed.,2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed.,1999, Academic Press; and the Oxford Dictionary Of Biochemistry AndMolecular Biology, Revised, 2000, Oxford University Press, provide oneof skill with a general dictionary of many of the terms used in thisdisclosure.

Units, prefixes, and symbols are denoted in their Systéme Internationalde Unites (SI) accepted form. Numeric ranges are inclusive of thenumbers defining the range. Unless otherwise indicated, amino acidsequences are written left to right in amino to carboxy orientation. Theheadings provided herein are not limitations of the various aspects orembodiments of the disclosure, which can be had by reference to thespecification as a whole. Accordingly, the terms defined immediatelybelow are more fully defined by reference to the specification in itsentirety. Amino acids are referred to herein by either their commonlyknown three letter symbols or by the one-letter symbols recommended bythe IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides,likewise, are referred to by their commonly accepted single-lettercodes.

The term “polypeptide,” “peptide” and “protein” are used interchangeablyand refer to a polymeric compound comprised of covalently linked aminoacid residues.

The term “polynucleotide” and “nucleic acid” are used interchangeablyand refer to a polymeric compound comprised of covalently linkednucleotide residues. Polynucleotides can be DNA, cDNA, RNA, singlestranded, or double stranded, vectors, plasmids, phage, or viruses.Polynucleotides include those in Table 10, which encode the polypeptidesof Table 11 (see Table 10). Polynucleotides also include fragments ofthe polynucleotides of Table 10, e.g., those that encode fragments ofthe polypeptides of Table 11, such as the Factor VIII, Fc, signalsequence, propeptide, and other fragments of the polypeptides of Table11.

“Administering,” as used herein, means to proscribe or give apharmaceutical composition comprising a Factor VIII polypeptide of theinvention to a subject via a pharmaceutically acceptable route. Examplesof routes of administration include, but are not limited to,intravenous, e.g., intravenous injection and intravenous infusion, e.g.,via central venous access. Additional routes of administration includesubcutaneous, intramuscular, oral, nasal, and pulmonary administration.Advantages of the present invention include: improved regimencompliance; reduced break through bleeds; increased protection of jointsfrom bleeds; prevention of joint damage; reduced morbidity; reducedmortality; prolonged protection from bleeding; decreased thromboticevents; and improved quality of life.

The terms “long-acting” and “long-lasting” are used interchangeablyherein. In one embodiment, the term “long-acting” or “long-lasting”indicates that a FVIII activity as a result of administration of the“long-acting” FVIII polypeptide is longer than the FVIII activity of awild-type FVIII (e.g., ADVATE® or plasma-derived FVIII (“pdFVIII”)). The“longer” FVIII activity can be measured by any known methods in the art,e.g., aPTT assay, chromogenic assay, ROTEM®, TGA, etc. In oneembodiment, the “longer” FVIII activity can be shown by the T_(1/2 beta)(activity). In another embodiment, the “longer” FVII activity can beshown by the level of FVIII antigen present in plasma, e.g., by theT_(1/2 beta) (antigen). In other embodiments, the long-acting orlong-lasting FVIII polypeptide works longer in a coagulation cascade,e.g., is active for a longer period, compared to a wild-type FVIIIpolypeptide, i.e., a polypeptide consisting of amino acids 20 to 1457 ofSEQ ID NO: 2, i.e., SQ BDD FVII (REFACTO®) or a polypeptide consistingof amino acids 20 to 2351 of SEQ ID NO: 6 (ADVATE®).

The terms “short-acting” and “short-lasting” are used interchangeablyherein. The term “short-acting” or “short-lasting” indicates that a FVIIactivity as a result of administration of the “short-acting” FVIIIpolypeptide is similar to or the same as the FVIII activity of awild-type FVIII (e.g., ADVATE® (amino acids 20 to 2351 of SEQ ID NO: 6),REFACTO® (amino acids 20 to 1457 of SEQ ID NO: 2), or plasma-derivedFVIII (“pdFVIII”)) or shorter than the FVIII activity of a long-actingFVIII polypeptide. The “shorter” FVIII activity can be measured by anyknown methods in the art, e.g., aPTT assay, chromogenic assay, ROTEM®,TGA, etc. Exemplary short-acting FVIII polypeptides include, but are notlimited to, wild-type mature FVIII polypeptide (ADVATE®, RECOMBINATE®,or HELIXATE®) or B-domain deleted FVIII polypeptides such as SQ BDDFVIII (REFACTO® and XYNTHA®) or FVIII polypeptides containing 21 aminoacids from B-domain (i.e., SFSQNSRHPSQNPPVLKRHQR, SEQ ID NO: 17) (e.g.,NOVOEIGHT®).

The term “chimeric polypeptide,” as used herein, means a polypeptidethat includes within it at least two polypeptides (or portions thereofsuch as subsequences or peptides) from different sources. Chimericpolypeptides can include two, three, four, five, six, seven, or morepolypeptides or portions thereof from different sources, such asdifferent genes, different cDNAs, or different animal or other species.Chimeric polypeptides can include one or more linkers joining thedifferent polypeptides or portions thereof. Thus, the polypeptides orportions thereof can be joined directly or they can be joinedindirectly, via linkers, or both, within a single chimeric polypeptide.Chimeric polypeptides can include additional peptides such as signalsequences and sequences such as 6His and FLAG that aid in proteinpurification or detection. In addition, chimeric polypeptides can haveamino acid or peptide additions to the N- and/or C-termini. In someembodiments, a chimeric polypeptide is a long-acting FVIII polypeptide.Exemplary chimeric polypeptides of the invention are Factor VIII-FcRn BPchimeric polypeptides, e.g., Factor VIII-Fc polypeptides such as theFVIIIFc, SEQ ID NO:2 or 6 (Table 11) with or without its signal sequenceand propeptide.

“Hybrid” polypeptides and proteins, as used herein, means a combinationof a chimeric polypeptide with a second polypeptide. The chimericpolypeptide and the second polypeptide in a hybrid can be associatedwith each other via protein-protein interactions, such as charge-chargeor hydrophobic interactions. The chimeric polypeptide and the secondpolypeptide in a hybrid can be associated with each other via disulfideor other covalent bond(s). Hybrids are described in WO 2004/101740 andWO 2006/074199, each of which is incorporated herein by reference in itsentirety. See also U.S. Pat. Nos. 7,404,956 and 7,348,004, each of whichis incorporated herein by reference in its entirety. The secondpolypeptide can be a second copy of the same chimeric polypeptide or itcan be a non-identical chimeric polypeptide. See, e.g., Table 11. In oneembodiment, the second polypeptide is a polypeptide comprising an FcRnbinding partner. FcRn binding partners binds to FcRn and protects theFcRn binding partner containing molecule from catabolism, thus extendingthe plasma half-life. In another embodiment, the chimeric polypeptide isa chimeric Factor VIII-Fc polypeptide and the second polypeptideconsists essentially of Fc, e.g., a rFVIIIFc recombinant fusion proteinconsisting of a single molecule of recombinant B-domain deleted humanFVIII (BDD-rFVIII) fused to the dimeric Fc domain of the human IgG1,with no intervening linker sequence. This hybrid polypeptide is referredto herein as FVIIIFc monomeric Fc fusion protein, FVILFc monomer hybrid,monomeric FVIIIFc hybrid, and FVIIIFc monomer-dimer. See Table 11A. TheExamples provide preclinical and clinical data for this hybridpolypeptide.

The second polypeptide in a hybrid can comprise or consist essentiallyof a sequence at least 90% or 95% identical to the amino acid sequenceshown in Table 11A(ii) without a signal sequence (amino acids 21 to 247of SEQ ID NO:4) or at least 90% or 95% identical to the amino acidsequence shown in Table 11A(ii) with a signal sequence (amino acids 1 to247 of SEQ ID NO:4). The second polypeptide can comprise or consistessentially of a sequence identical to the amino acid sequence shown inTable 11A(ii) without a signal sequence (amino acids 21 to 247 of SEQ IDNO:4) or identical to the amino acid sequence shown in Table 11A(ii)with a signal sequence (amino acids 1 to 247 of SEQ ID NO:4).

Factor VIII coagulant activity is expresses as International Unit(s)(IU). One IU of Factor VIII activity corresponds approximately to thequantity of Factor VIII in one milliliter of normal human plasma.Several assays are available for measuring Factor VII activity,including the one stage clotting assay (activated partial thromboplastintime; aPTT), thrombin generation time (TGA) and rotationalthromboelastometry (ROTEM®).

The term “lyophilisate” as used herein in connection with theformulation according to the invention denotes a formulation which ismanufactured by freeze-drying methods known in the art per se. Thesolvent (e.g. water) is removed by freezing following sublimation undervacuum and desorption of residual water at elevated temperature. In thepharmaceutical field, the lyophilisate has usually a residual moistureof about 0.1 to 5% (w/w) and is present as a powder or a physical stablecake. The lyophilisate is characterized by a fast dissolution afteraddition of a reconstitution medium.

The term “reconstituted formulation” as used herein denotes aformulation which is lyophilized and re-dissolved by addition of adiluent. The diluent can contain, without limitation, water forinjection (WFI), bacteriostatic water for injection (BWFI), sodiumchloride solutions (e.g. 0.9% (w/v) NaCl), glucose solutions (e.g. 5^(%)glucose), surfactant containing solutions (e.g. 0.01% polysorbate 20 orpolysorbate 80), a pH-buffered solution (e.g. phosphate-bufferedsolutions) and combinations thereof.

“Dosing interval,” as used herein, means the amount of time that elapsesbetween multiple doses being administered to a subject. Dosing intervalcan thus be indicated as ranges. The dosing interval in the methods ofthe invention using a chimeric FVIII-FcRn BP, e.g., a chimeric FVIII-Fc,can be at least about one and one-half to eight times longer than thedosing interval required for an equivalent amount (in IU/kg) of theFactor VIII without the FcRn BP, e.g., Fc portion (i.e., a polypeptideconsisting of said FVIII). The dosing interval when administering, e.g.,a Factor VIII-Fc chimeric polypeptide (or a hybrid) of the invention canbe at least about one and one-half times longer than the dosing intervalrequired for an equivalent amount of the Factor VII without the FcRn BP,e.g., Fc portion (i.e., a polypeptide consisting of the Factor VIII).The dosing interval can be at least about one and one-half to eighttimes longer than the dosing interval required for an equivalent amountof the Factor VIII without, e.g., the Fc portion (or a polypeptideconsisting of the Factor VIII).

The term “dosing frequency” as used herein refers to the frequency ofadministering doses of a FVII polypeptide in a given time. Dosingfrequency can be indicated as the number of doses per a given time,e.g., once a week or once in two weeks.

The term “prophylaxis of one or more bleeding episode” or “prophylactictreatment” as used herein means administering a FVIII polypeptide inmultiple doses to a subject over a course of time to increase the levelof FVIII activity in a subject's plasma. In one embodiment, “prophylaxisof one or more bleeding episode” indicates use of a FVIII polypeptide toprevent or inhibit occurrence of one or more spontaneous oruncontrollable bleeding or bleeding episodes or to reduce the frequencyof one or more spontaneous or uncontrollable bleeding or bleedingepisodes. In another embodiment, the increased FVIII activity level issufficient to decrease the incidence of spontaneous bleeding or toprevent bleeding in the event of an unforeseen injury. Prophylactictreatment decreases or prevents bleeding episodes, for example, thosedescribed under on-demand treatment. Prophylactic treatment can beindividualized, as discussed under “dosing interval”, e.g., tocompensate for inter-patient variability.

The term “about twice weekly” as used herein means approximate number,and “about twice weekly” can include twice in one week, e.g., a firstdose in three days and a second dose in three days, a first dose inthree days and a second dose in four days, a first dose in four days anda second dose in three days, a first dose in four days and a second dosein four days. The term “about twice weekly” can also include every threedays, every four days, or every five days.

The term “about once a week” as used herein means approximate number,and “about once a week” can include every seven days±two days, i.e.,every five days to every nine days. The dosing frequency of “once aweek” thus can be every five days, every six days, every seven days,every eight days, or every nine days.

The term “individualized prophylaxis” or “prophylactic andindividualized” as used herein means use of a FVIII polypeptide for anindividualized dosing and/or dosing interval or frequency to prevent orinhibit occurrence of one or more spontaneous and/or uncontrollablebleeding or bleeding episodes or to reduce the frequency of one or morespontaneous and/or uncontrollable bleeding or bleeding episodes.“Individualized” within the context of prophylaxis is used synonymouswith “tailored” throughout this application. For example,“individualized prophylaxis” also means “tailored prophylaxis” and“prophylactic and individualized” also means “prophylactic andtailored.” In one embodiment, the “individualized interval” includesevery 3 days+2 days, i.e., every day to every five days. The dosingfrequency of the “individualized interval prophylaxis” thus can be everyday, every two days, every three days, every four days, or every fivedays.

The term “on-demand treatment,” as used herein, means treatment that isintended to take place over a short course of time and is in response toan existing condition, such as a bleeding episode, or a perceived shortterm need such as planned surgery. The “on-demand treatment” is usedinterchangeably with “episodic” treatment. Conditions that can requireon-demand treatment include a bleeding episode, hemarthrosis, musclebleed, oral bleed, hemorrhage, hemorrhage into muscles, oral hemorrhage,trauma, trauma capitis, gastrointestinal bleeding, intracranialhemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, bonefracture, central nervous system bleeding, bleeding in theretropharyngeal space, bleeding in the retroperitoneal space, orbleeding in the illiopsoas sheath. Bleeding episodes other than theseare also included. The subject can be in need of surgical prophylaxis,peri-operative management, or treatment for surgery. Such surgeriesinclude minor surgery, major surgery, tooth extraction, tonsillectomy,other dental/thoraco-facial surgeries, inguinal herniotomy, synovectomy,total knee replacement, other joint replacement, craniotomy,osteosynthesis, trauma surgery, intracranial surgery, intra-abdominalsurgery, or intrathoracic surgery. Surgeries other than these are alsoincluded.

Additional conditions that can require on-demand treatment include minorhemorrhage, hemarthroses, superficial muscle hemorrhage, soft tissuehemorrhage, moderate hemorrhage, intramuscle or soft tissue hemorrhagewith dissection, mucous membrane hemorrhage, hematuria, majorhemorrhage, hemorrhage of the pharynx, hemorrhage of the retropharynx,hemorrhage of the retroperitonium, hemorrhage of the central nervoussystem, bruises, cuts, scrapes, joint hemorrhage, nose bleed, mouthbleed, gum bleed, intracranial bleeding, intraperitoneal bleeding, minorspontaneous hemorrhage, bleeding after major trauma, moderate skinbruising, or spontaneous hemorrhage into joints, muscles, internalorgans or the brain. Additional reasons for on-demand treatment includethe need for peri-operative management for surgery or dental extraction,major surgery, extensive oral surgery, urologic surgery, hernia surgery,orthopedic surgery such as replacement of knee, hip, or other majorjoint.

The term “treatment” or “treating” as used herein means amelioration orreduction of one or more symptoms of bleeding diseases or disordersincluding, but not limited to, hemophilia B. In one embodiment,“treatment of” or “treating” a bleeding disease or disorder includesprevention of one or more symptoms of a bleeding disease or disorder. Ina bleeding disease or disorder caused by a FVIII deficiency (e.g., a lowbaseline FVIII activity), the term “treatment” or “treating” means aFVIII replacement therapy. By administering a pharmaceutical compositioncomprising a FVIII polypeptide to a subject, the subject can achieveand/or maintain a plasma trough level of a FVIII activity at about 1IU/dl or above 1 IU/dl. In other embodiments, “treatment” or “treating”means reduction of the frequency of one or more symptoms of bleedingdiseases or disorders, e.g., spontaneous or uncontrollable bleedingepisodes. “Treatment,” however, need not be a cure.

The term “perioperative management” as used herein means use of apharmaceutical composition comprising a FVII polypeptide before,concurrently with, or after an operative procedure, e.g., a surgicaloperation. The use for “perioperative management” of one or morebleeding episode includes surgical prophylaxis before (i.e.,preoperative), during (i.e., intraoperative), or after (i.e.,postoperative) a surgery to prevent one or more bleeding or bleedingepisode or reducing or inhibiting spontaneous and/or uncontrollablebleeding episodes before, during, and after a surgery.

“Baseline,” as used herein, is the lowest measured plasma Factor VIIIlevel in a subject prior to administering a dose. The FVIII plasmalevels can be measured at two time points prior to dosing: at ascreening visit and immediately prior to dosing. Alternatively, (a) thebaseline in patients whose pretreatment FVII activity is <1%, who haveno detectable FVI antigen, and have nonsense genotypes can be defined as0%, (b) the baseline for patients with pretreatment FVIII activity <1%and who have detectable FVII antigen can be set at 0.5%, (c) thebaseline for patients whose pretreatment FVIII activity is between 1-2%is Cmin (the lowest activity throughout the PK study), and (d) thebaseline for patients whose pretreatment FVIII activity is ≥2% can beset at 2%. Activity above the baseline pre-dosing can be consideredresidue drug from prior treatment, and can be decayed to baseline andsubtracted from the PK data following rFVIIIFc dosing.

“T_(1/2β),” or “T_(1/2 beta)” or “Beta HL,” as used herein, is half-lifeassociated with elimination phase, t_(1/2β)=(ln 2)/elimination rateconstant associated with the terminal phase. The T_(1/2 beta) can bemeasured by FVIII activity or by FVII antigen level in plasma. TheT_(1/2 beta) based on activity is shown as T_(1/2 beta) (activity), andthe Tin b based on the FVIII antigen level can be shown as TT_(1/2 beta)(antigen). Both TT_(1/2 beta) (activity) and TT_(1/2 beta) (antigen) canbe shown as ranges or a geometric mean.

“Trough,” as used herein, is the lowest plasma FVIII activity levelreached after administering a dose of chimeric polypeptide of theinvention or another FVII molecule and before the next dose isadministered, if any. Trough is used interchangeably herein with“threshold.” Baseline FVIII levels are subtracted from measured FVIIIlevels to calculate the trough level.

The term “annualized bleeding rate” (“ABR”) as used herein refers to thenumber of bleeding episodes (including spontaneous and traumatic bleeds)experienced by a subject during a defined time period, extrapolated to 1year. For example two bleeds in six months would indicate an ABR offour. The median ABR provides the middle value among all observed ABRs,indicating that half of the subjects had individual ABRs less than orequal to the median and half had ABRs greater than or equal to themedian.

The term “inter quartile range” (“IQR”) as used herein refers to ameasure of statistical dispersion, being equal to the difference betweenthe upper and lower quartiles. Unlike (total) range, the interquartilerange is a robust statistic, having a breakdown point of 25%, and isthus often preferred to the total range. For a symmetric distribution(where the median equals the midline, the average of the first and thirdquartiles), half the IQR equals the median absolute deviation (MAD). Themedian is the corresponding measure of central tendency.

“Subject,” as used herein means a human. Subject as used herein includesan individual who is known to have at least one incidence ofuncontrolled bleeding episodes, who has been diagnosed with a disease ordisorder associated with uncontrolled bleeding episodes, e.g., ableeding disease or disorder, e.g., hemophilia A, who is susceptible touncontrolled bleeding episodes, e.g., hemophilia, or any combinationsthereof. Subjects can also include an individual who is in danger of oneor more uncontrollable bleeding episodes prior to a certain activity,e.g., a surgery, a sport activity, or any strenuous activities. Thesubject can have a baseline FVIII activity less than 1%, less than 0.5%,less than 2%, less than 2.5%, less than 3%, or less than 4%. Subjectsalso include pediatric humans. Pediatric human subjects are birth to 20years, preferably birth to 18 years, birth to 16 years, birth to 15years, birth to 12 years, birth to 11 years, birth to 6 years, birth to5 years, birth to 2 years, or 2 to 11 years of age.

“Therapeutic dose,” “dose,” “effective dose,” or “dosing amount” as usedherein, means a dose that achieves a plasma trough level of a FVIIIactivity at least about 1 IU/dl or above 1 IU/dl in the subjectadministered with a pharmaceutical composition comprising a FVIIIpolypeptide. For the purpose of this invention, in one embodiment, the“dose” refers to the amount of the doses that a plasma trough level of aFVIII activity is maintained at least about 1 IU/dl or above 1 IU/dl, atleast about 2 IU/dl or above 2 IU/dl, at least about 3 IU/dl or above 3IU/dl, at least about 4 IU/dl or above 4 IU/dl, or at least about 5IU/dl or above 5 IU/dl throughout the administration of a pharmaceuticalcomposition comprising a FVIII polypeptide. In another embodiment, the“dose” reduces or decreases frequency of bleeding or bleeding disorder.In other embodiments, the “dose” stops on-going, uncontrollable bleedingor bleeding episodes. In still other embodiments, the “dose” preventsspontaneous bleeding or bleeding episodes in a subject susceptible tosuch spontaneous bleeding or bleeding episodes. The “dose” or“therapeutic dose” need not cure hemophilia.

The term “target joint” is defined as a major joint (e.g., hip, elbow,wrist, shoulder, knee, and ankle) into which repeated bleeding occurs(frequency of 23 bleeding episodes into the same joint in a consecutive6-month period).

The term “bleeding episode” as used herein adopts a standardizeddefinition of a bleeding episode. A bleeding episode started from thefirst sign of bleeding, and ended 72 hours after the last treatment forthe bleeding, within which any symptoms of bleeding at the samelocation, or injections less than or equal to 72 hours apart, wereconsidered the same bleeding episode. Any injection to treat thebleeding episode, taken more than 72 hours after the preceding one, wasconsidered the first injection to treat a new bleeding episode at thesame location. Any bleeding at a different location was considered aseparate bleeding episode regardless of time from last injection. Thisdefinition has been proposed by the Subcommittee on Standards andCriteria, FVIII/FIX subcommittee of the International Society ofThrombosis and Hemostasis and has been used by the PedNet multicenterstudy in hemophilia.

The term “annualized bleeding rates (ABRs) as used herein refers to thenumber of bleeding episodes that are annualized for each patient usingthe following formula:

${ABR} = {\frac{{Number}\mspace{14mu} {of}\mspace{14mu} {bleeding}\mspace{14mu} {episodes}\mspace{14mu} {during}\mspace{14mu} {efficacy}\mspace{14mu} {period}}{{Total}\mspace{14mu} {number}\mspace{14mu} {of}\mspace{14mu} {days}\mspace{14mu} {during}\mspace{14mu} {the}\mspace{14mu} {efficacy}\mspace{14mu} {period}} \times 365.25}$

“Variant,” as used herein, refers to a polynucleotide or polypeptidediffering from the original polynucleotide or polypeptide, but retainingessential properties thereof, e.g., Factor VIII coagulant activity or Fc(FcRn binding) activity. Generally, variants are overall closelysimilar, and, in many regions, identical to the original polynucleotideor polypeptide. Variants include polypeptide and polynucleotidefragments, deletions, insertions, and modified versions of originalpolypeptides.

II. Pharmaceutical Composition

The present invention is directed to a FVIII polypeptide formulated as apharmaceutical composition. The pharmaceutical composition can beformulated for administration to humans. The FVIII polypeptide can be ashort-acting FVIII polypeptide or a long-acting FVIII polypeptide. Thepharmaceutical compositions comprise pharmaceutically acceptablecarriers, including, e.g., ion exchangers, alumina, aluminum stearate,lecithin, serum proteins, such as human serum albumin, buffer substancessuch as phosphates, glycine, sorbic acid, potassium sorbate, partialglyceride mixtures of saturated vegetable fatty acids, water, salts orelectrolytes, such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-basedsubstances, polyethylene glycol, sodium carboxymethylcellulose,polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers,polyethylene glycol and wool fat. Various methods of formulating theinvention are well known in the art.

In certain formulations provided herein, a FVIII polypeptide isformulated as a sterile, preservative-free, non-pyrogenic, lyophilized,white to off-white powder to cake, for intravenous (IV) administration.The formulation can be provided in a single-use vial.

In certain embodiments, a FVIII polypeptide (e.g., a long-acting orshort-acting FVIII polypeptide, e.g., rFVIIIFc) formulation is providedin a single-use vial manufactured to contain, following reconstitutionwith an appropriate amount of diluent, about 83 IU/ml, 167 IU/ml, 250IU/ml, 333 IU/ml, 500 IU/ml, 667 IU/ml, 1000 IU/ml, 1333 IU/ml, 1667IU/ml, or 2000 IU/ml of the long-acting FVIII polypeptide. In certainembodiments in which diluent is added to a final volume of about 3 ml, asingle-use vial can nominally contain about 250, about 500, about 750,about 1000, about 1500, about 2000, about 3000, about 4000, about 5000,or about 6000 International Units (IU) of the FVIII polypeptide (e.g., along-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc).

In certain embodiments the formulation includes, in addition to theactive FVIII polypeptide (e.g., a long-acting or short-acting FVIIIpolypeptide, e.g., rFVIIIFc): sucrose (which can act as a stabilizer),sodium chloride (which can act as a bulking agent), L-histidine (whichcan act as a buffer), calcium chloride, and polysorbate 20 orpolysorbate 80 (which can act as a stabilizer). The formulation isprovided with a diluent comprising a sterile sodium chloride solution.In certain embodiments, the diluent is provided in a pre-filled syringe.

Accordingly, provided herein is a pharmaceutical composition comprisinga specified amount of a FVIII polypeptide (e.g., a long-acting orshort-acting FVIII polypeptide, e.g., rFVIIIFc) (in IU), along with theexcipients sucrose, NaCl, L-histidine, calcium chloride, and polysorbate20 or polysorbate 80. The compositions provided herein comprise variousconcentrations of the various excipients, and the concentrations can beexpressed in various ways. For example, the concentration of a givenexcipient can be expressed as a molar concentration (e.g., M or mM), asa weight/volume percent, (e.g., grams per 100 ml diluent), or asmilligrams per milliliter (mg/ml). Formulations provided herein cancontain specified amounts of the various excipients at a level ofprecision ranging from approximate, e.g., concentrations expressed onlyto one significant figure (e.g., about 0.1% (w/v)), or with moreprecision, e.g., out to 2, 3, 4, 5, or 6 significant figures (e.g.,about 3.88 mg/ml, with precision out to three significant figures). Thenecessary level of precision can vary depending on, e.g., therequirements of a given regulatory agency, or the manufacturing process.In certain embodiments the pharmaceutical composition comprises areconstituted formulation, which can be provided as a lyophilisate,optionally accompanied by a diluent.

In certain embodiments, the pharmaceutical composition comprises about50 IU/ml to about 2500 IU/ml rFVIIIFc, e.g., 83 IU/ml, 167 IU/ml, 250IU/ml, 333 IU/ml, 500 IU/ml, 667 IU/ml, 1000 IU/ml, 1333 IU/ml, 1667IU/ml, or 2000 IU/ml of a FVIII polypeptide (e.g., a long-acting orshort-acting FVIII polypeptide, e.g., rFVIIIFc). In certain embodiments,the pharmaceutical composition comprises 83 IU/ml, 167 IU/ml, 250 IU/ml,333 IU/ml, 500 IU/ml, 667 IU/ml, 1000 IU/ml, 1333 IU/ml, 1667 IU/ml, or2000 IU/ml of a FVIII polypeptide (e.g., a long-acting or short-actingFVIII polypeptide, e.g., rFVIIIFc) in a formulation comprising about13.3 mg/ml or about 1.33% (w/v) sucrose, about 12.0 mg/ml or about 205mM NaCl, about 1.03 mg/ml or about 6.64 mM L-histidine, about 0.80 mg/mlor about 5.4 mM calcium chloride dihydrate, and about 0.13 mg/ml orabout 0.013% (w/v) polysorbate 20 or polysorbate 80.

In certain embodiments, the pre-lyophilization pharmaceuticalcomposition comprises about 100 IU/ml to about 4000 IU/ml rFVIIIFc,e.g., 150 IU/ml, 287.5 IU/ml, 431.25 IU/ml, 575 IU/ml, 862.5 IU/ml, 1150IU/ml, 1725 IU/ml, 2300 IU/ml, 2875 IU/ml, or 3450 IU/ml of a FVIIIpolypeptide (e.g., a long-acting or short-acting FVIII polypeptide,e.g., rFVIIIFc). In certain embodiments, the pharmaceutical compositioncomprises 150 IU/mi, 287.5 IU/ml, 431.25 IU/ml, 575 IU/ml, 862.5 IU/ml,1150 IU/ml, 1725 IU/ml, 2300 IU/ml, 2875 IU/ml, or 3450 IU/ml of a FVIIIpolypeptide (e.g., a long-acting or short-acting FVIII polypeptide,e.g., rFVIIIFc) in a formulation comprising about 20.0 mg/ml or about2.0% (w/v) sucrose, about 18.0 mg/ml or about 308 mM NaCl, about 1.55mg/ml or about 9.8 mM L-histidine, about 1.18 mg/ml or about 8.0 mMcalcium chloride dihydrate, and about 0.2 mg/ml or about 0.02% (w/v)polysorbate 20 or polysorbate 80.

In certain embodiments, the pharmaceutical composition comprises apharmaceutically acceptable amount of sucrose. In certain embodiments,the pharmaceutical composition comprises about 1% (w/v) to about 2.5%(w/v) sucrose, preferably about 1.3% (w/v) to about 2% sucrose (w/v)e.g., about 1.33% (w/v) sucrose or about 2.0% (w/v) sucrose. In certainrelated embodiments the pharmaceutical composition comprises about 10mg/ml to about 25 mg/ml sucrose, preferably about 13 mg/ml to about 20mg/ml sucrose, e.g., about 13.3 mg/ml sucrose or about 20.0 mg/mlsucrose. In still other embodiments, stabilizing agents such astrehalose, raffinose and/or arginine can be used at these concentrationsin lieu of or in combination with sucrose.

In certain embodiments, the pharmaceutical composition comprises about150 mM to about 250 mM NaCl, about 175 mM to about 225 mM NaCl, andabout 200 mM to about 210 mM NaCl, e.g., about 205 mM NaCl. In certainrelated embodiments, the pharmaceutical composition comprises about 8.8mg/ml to about 14.6 mg/ml NaCl, about 10 mg/ml NaCl to about 13 mg/ml,e.g., about 12.0 mg/ml NaCl. In certain embodiments, NaCl is provided atthe desired concentration in a diluent solution in which a lyophilisatecomprising a FVIII polypeptide (e.g., a long-acting or short-actingFVIII polypeptide, e.g., rFVIIIFc) is reconstituted.

In another embodiment, the compositions of the invention do not includebulking agents such as mannitol, glycine, alanine and/or hydroxyethylstarch. In other embodiments, NaCl is the sole bulking agent.

In certain embodiments, the pre-lyophilization pharmaceuticalcomposition comprises about 250 mM to about 350 mM NaCl, about 275 mM toabout 325 mM NaCl, e.g., about 308 mM NaCl. In certain relatedembodiments, the pharmaceutical composition comprises about 14.6 mg/mlto about 20.5 mg/ml NaCl, about 16 mg/ml to about 19 mg/ml, e.g., about18.0 mg/ml NaCl.

In certain embodiments, the pharmaceutical composition comprises apharmaceutically acceptable amount of L-histidine. In certainembodiments, the pharmaceutical composition comprises about 5 mM toabout 15 mM L-histidine, e.g., about 6.64 mM L-histidine or about 9.8 mML-histidine. In certain related embodiments the pharmaceuticalcomposition comprises about 0.75 mg/ml to about 2.25 mg/ml L-histidine,e.g., about 1.03 mg/ml L-histidine or about 1.55 mg/ml L-histidine. Incertain embodiments, L-histidine is provided as part of a lyophilisate,which, upon reconstitution with an appropriate amount of diluentprovides L-histidine at the desired concentration.

In certain embodiments, the pharmaceutical composition comprises apharmaceutically acceptable amount of calcium chloride. In certainembodiments, the pharmaceutical composition comprises about 5 mM toabout 10 mM calcium chloride, e.g., about 5.4 mM calcium chloride orabout 8 mM calcium chloride. In certain related embodiments thepharmaceutical composition comprises about 0.75 mg/ml to about 1.5 mg/mlcalcium chloride dihydrate, e.g., about 0.8 mg/ml calcium chloridedihydrate or about 1.18 mg/ml calcium chloride dihydrate. In certainembodiments, calcium chloride is provided as part of a lyophilisate,which, upon reconstitution with an appropriate amount of diluentprovides calcium chloride at the desired concentration.

In certain embodiments, the pharmaceutical composition comprises apharmaceutically acceptable amount of polysorbate 20 or polysorbate 80.In certain related embodiments the pharmaceutical composition comprisesabout 0.008% (w/v) to about 0.025% (w/v) polysorbate 20 or polysorbate80, e.g., about 0.013% (w/v) polysorbate 20 or polysorbate 80 or about0.02% (w/v) polysorbate 20 or polysorbate 80. In certain relatedembodiments the pharmaceutical composition comprises about 0.08 mg/ml toabout 0.25 mg/ml polysorbate 20 or polysorbate 80, e.g., about 0.13%mg/ml polysorbate 20 or polysorbate 80 or about 0.20 mg/ml polysorbate20 or polysorbate 80. In certain embodiments, polysorbate 20 orpolysorbate 80 is provided as part of a lyophilisate, which, uponreconstitution with an appropriate amount of diluent providespolysorbate 20 or polysorbate 80 at the desired concentration.

In certain embodiments, the pharmaceutical composition comprises: about50 IU/m to about 2500 IU/ml of a FVIII polypeptide (e.g., a long-actingor short-acting FVIII polypeptide, e.g., rFVIIIFc); about 1% (w/v) toabout 2.5% (w/v) of sucrose; about 150 mM to about 250 mM NaCl; about 5mM to about 15 mM L-histidine; about 5 mM to about 10 mM calciumchloride; and about 0.008% (w/v) to about 0.025% of polysorbate 20 orpolysorbate 80. In certain embodiments the pharmaceutical composition isprovided as a lyophilisate and a diluent. In certain embodiments theamount of lyophilizate provides about 3 ml of a pharmaceuticalcomposition with the desired ingredients at the desired concentrations.

In certain embodiments, the pharmaceutical composition comprises: about50 IU/m to about 2500 IU/ml of a FVIII polypeptide (e.g., a long-actingor short-acting FVIII polypeptide, e.g., rFVIIIFc); about 10 mg/ml toabout 25 mg/ml of sucrose; about 8.8 mg/ml to about 14.6 mg/ml NaCl;about 0.75 mg/ml to about 2.25 mg/ml L-histidine; about 0.75 mg/ml toabout 1.5 mg/ml calcium chloride dihydrate; and about 0.08 mg/ml toabout 0.25 mg/ml of polysorbate 20 or polysorbate 80. In certainembodiments the pharmaceutical composition is provided as a lyophilisateand a diluent. In certain embodiments the amount of lyophilizateprovides about 3 ml of a pharmaceutical composition with the desiredingredients at the desired concentrations.

Exemplary compositions are provided in Table 1 and in Table 2 in theExamples.

For example, the disclosure provides a pharmaceutical compositioncomprising: about 83 IU/ml of a FVIII polypeptide (e.g., a long-actingor short-acting FVIII polypeptide, e.g., rFVIIIFc); about 1.33% (w/v) ofsucrose; about 205 mM NaCl; about 6.64 mM L-histidine; about 5.4 mMcalcium chloride dihydrate, and about 0.013% (w/v) of polysorbate 20 orpolysorbate 80. The disclosure further provides a pharmaceuticalcomposition comprising: about 167 IU/ml of a FVIII polypeptide (e.g., along-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc); about1.33% (w/v) of sucrose; about 205 mM NaCl; about 6.64 mM L-histidine;about 5.4 mM calcium chloride dihydrate, and about 0.013% (w/v) ofpolysorbate 20 or polysorbate 80. The disclosure further provides apharmaceutical composition comprising: about 250 IU/ml of a FVIIIpolypeptide (e.g., a long-acting or short-acting FVIII polypeptide,e.g., rFVIIIFc); about 1.33% (w/v) of sucrose; about 205 mM NaCl; about6.64 mM L-histidine; about 5.4 mM calcium chloride dihydrate, and about0.013% (w/v) of polysorbate 20 or polysorbate 80. The disclosure furtherprovides a pharmaceutical composition comprising: about 333 IU/ml of aFVIII polypeptide (e.g., a long-acting or short-acting FVIIIpolypeptide, e.g., rFVIIIFc); about 1.33% (w/v) of sucrose; about 205 mMNaCl; about 6.64 mM L-histidine; about 5.4 mM calcium chloridedihydrate, and about 0.013% (w/v) of polysorbate 20 or polysorbate 80.The disclosure further provides a pharmaceutical composition comprising:about 500 IU/ml of a FVII polypeptide (e.g., a long-acting orshort-acting FVIII polypeptide, e.g., rFVIIIFc); about 1.33% (w/v) ofsucrose; about 205 mM NaCl; about 6.64 mM L-histidine; about 5.4 mMcalcium chloride dihydrate, and about 0.013% (w/v) of polysorbate 20 orpolysorbate 80. The disclosure further provides a pharmaceuticalcomposition comprising: about 667 IU/ml of a FVIII polypeptide (e.g., along-acting or short-acting FVII polypeptide, e.g., rFVIIIFc); about1.33% (w/v) of sucrose; about 205 mM NaCl; about 6.64 mM L-histidine;about 5.4 mM calcium chloride dihydrate, and about 0.013% (w/v) ofpolysorbate 20 or polysorbate 80. The disclosure further provides apharmaceutical composition comprising: about 1000 IU/ml of a FVHIIIpolypeptide (e.g., a long-acting or short-acting FVIII polypeptide,e.g., rFVIIIFc); about 1.33% (w/v) of sucrose; about 205 mM NaCl; about6.64 mM L-histidine; about 5.4 mM calcium chloride, and about 0.013%(w/v) of polysorbate 20 or polysorbate 80. The disclosure also providesa pharmaceutical composition comprising: about 1333 IU/ml of a FVIIIpolypeptide (e.g., a long-acting or short-acting FVIII polypeptide,e.g., rFVIIFc); about 1.33% (w/v) of sucrose; about 205 mM NaCl; about6.64 mM L-histidine; about 5.4 mM calcium chloride, and about 0.013%(w/v) of polysorbate 20 or polysorbate 80. In some embodiments, thedisclosure provides a pharmaceutical composition comprising: about 1667IU/ml of a FVIII polypeptide (e.g., a long-acting or short-acting FVIIpolypeptide, e.g., rFVIIIFc); about 1.33% (w/v) of sucrose; about 205 mMNaCl; about 6.64 mM L-histidine; about 5.4 mM calcium chloride, andabout 0.013% (w/v) of polysorbate 20 or polysorbate 80. In otherembodiments, the disclosure provides a pharmaceutical compositioncomprising: about 2000 IU/ml of a FVIII polypeptide (e.g., a long-actingor short-acting FVIII polypeptide, e.g., rFVIIIFc); about 1.33% (w/v) ofsucrose; about 205 mM NaCl; about 6.64 mM L-histidine; about 5.4 mMcalcium chloride, and about 0.013% (w/v) of polysorbate 20 orpolysorbate 80.

The disclosure further provides a pharmaceutical composition comprising:about 83 IU/ml of a FVIII polypeptide (e.g., a long-acting orshort-acting FVIII polypeptide, e.g., rFVIIIFc); about 13.3 mg/ml ofsucrose; about 12.0 mg/ml NaCl; about 1.03 mg/ml L-histidine; about 0.80mg/ml calcium chloride dihydrate, and about 0.10 mg/ml of polysorbate 20or polysorbate 80. The disclosure further provides a pharmaceuticalcomposition comprising: about 1673 IU/mi of a FVIII polypeptide (e.g., along-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc); about13.3 mg/ml of sucrose; about 12.0 mg/ml NaCl; about 1.03 mg/mlL-histidine; about 0.80 mg/ml calcium chloride dihydrate, and about 0.10mg/ml of polysorbate 20 or polysorbate 80. The disclosure furtherprovides a pharmaceutical composition comprising: about 250 IU/ml of aFVIII polypeptide (e.g., a long-acting or short-acting FVIIIpolypeptide, e.g., rFVIIIFc); about 13.3 mg/ml of sucrose; about 12.0mg/ml NaCl; about 1.03 mg/ml L-histidine; about 0.80 mg/ml calciumchloride dihydrate, and about 0.10 mg/ml of polysorbate 20 orpolysorbate 80. The disclosure further provides a pharmaceuticalcomposition comprising: about 333 IU/ml of a FVIII polypeptide (e.g., along-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc); about13.3 mg/ml of sucrose; about 12.0 mg/ml NaCl; about 1.03 mg/mlL-histidine; about 0.80 mg/ml calcium chloride dihydrate, and about 0.10mg/ml of polysorbate 20 or polysorbate 80. The disclosure furtherprovides a pharmaceutical composition comprising: about 500 IU/ml of aFVIII polypeptide (e.g., a long-acting or short-acting FVIIIpolypeptide, e.g., rFVIIIFc); about 13.3 mg/ml of sucrose; about 12.0mg/ml NaCl; about 1.03 mg/ml L-histidine; about 0.80 mg/ml calciumchloride dihydrate, and about 0.10 mg/ml of polysorbate 20 orpolysorbate 80. The disclosure further provides a pharmaceuticalcomposition comprising: about 667 IU/ml of a FVIII polypeptide (e.g., along-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc); about13.3 mg/ml of sucrose; about 12.0 mg/ml NaCl; about 1.03 mg/mlL-histidine; about 0.80 mg/ml calcium chloride dihydrate, and about 0.10mg/ml of polysorbate 20 or polysorbate 80. The disclosure furtherprovides a pharmaceutical composition comprising: about 1000 IU/ml of aFVII polypeptide (e.g., a long-acting or short-acting FVIII polypeptide,e.g., rFVIIIFc); about 13.3 mg/ml of sucrose; about 12.0 mg/ml NaCl;about 1.03 mg/ml L-histidine; about 0.80 mg/ml calcium chloridedihydrate, and about 0.10 mg/ml of polysorbate 20 or polysorbate 80. Thedisclosure also provides a pharmaceutical composition comprising: about1333 IU/ml of a FVIII polypeptide (e.g., a long-acting or short-actingFVIII polypeptide, e.g., rFVIIIFc); about 13.3 mg/ml of sucrose; about12.0 mg/ml NaCl; about 1.03 mg/ml L-histidine; about 0.80 mg/ml calciumchloride dihydrate, and about 0.10 mg/ml of polysorbate 20 orpolysorbate 80. In some embodiments, the disclosure provides apharmaceutical composition comprising: about 1667 IU/ml of a FVIIIpolypeptide (e.g., a long-acting or short-acting FVII polypeptide, e.g.,rFVIIIFc); about 13.3 mg/ml of sucrose; about 12.0 mg/ml NaCl; about1.03 mg/ml L-histidine; about 0.80 mg/ml calcium chloride dihydrate, andabout 0.10 mg/ml of polysorbate 20 or polysorbate 80. In otherembodiments, the disclosure provides a pharmaceutical compositioncomprising: about 2000 IU/ml of a FVIII polypeptide (e.g., a long-actingor short-acting FVIII polypeptide, e.g., rFVIIIFc); about 13.3 mg/ml ofsucrose; about 12.0 mg/ml NaCl; about 1.03 mg/ml L-histidine; about 0.80mg/ml calcium chloride dihydrate, and about 0.10 mg/ml of polysorbate 20or polysorbate 80.

This disclosure also provides the components of a pharmaceutical kit.Such a kit includes one or more containers and optional attachments. Akit as provided herein facilitates administration of an effective amountof the FVIII polypeptide (e.g., a long-acting or short-acting FVIIIpolypeptide, e.g., rFVIIIFc) to a subject in need thereof. In certainembodiments, the kit facilitates administration of the FVIII polypeptide(e.g., a long-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc)via intravenous infusion. In certain embodiments, the kit facilitatesself-administration of the FVII polypeptide (e.g., a long-acting orshort-acting FVIII polypeptide, e.g., rFVIIIFc) via intravenousinfusion.

In certain embodiments, the disclosure provides a pharmaceutical kitcomprising: a first container comprising a lyophilized powder or cake,where the powder or cake comprises: (i) a FVIII polypeptide (e.g., along-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc), (ii)sucrose (and/or trehalose, raffinose or arginine); (iii) NaCl; (iv)L-histidine; (v) calcium chloride dihydrate; and (vi) polysorbate 20 orpolysorbate 80; and a second container comprising sterilized water forinjections to be combined with the lyophilized powder of the firstcontainer. In certain embodiments, sufficient diluent is provided toproduce about 3 ml of a FVII polypeptide (e.g., a long-acting orshort-acting FVIII polypeptide, e.g., rFVIIIFc) formulation with desiredproperties as disclosed herein. In certain embodiments, the secondcontainer is a pre-filled syringe associated with a plunger, to allowaddition of the diluent to the first container, reconstitution of thecontents of the first container, and transfer back into the syringe. Incertain embodiments, the kit further provides an adaptor for attachingthe syringe to the first container. In certain embodiments the kitfurther provides a needle and infusion tubing, to be attached to thesyringe containing the reconstituted FVIII polypeptide (e.g., along-acting or short-acting FVII polypeptide, e.g., rFVIIIFc)formulation to allow IV infusion of the formulation.

In certain embodiments a FVIII polypeptide (e.g., a long-acting orshort-acting FVIII polypeptide, e.g., rFVIIIFc) is provided in a totalamount from about 200 IU to about 6000 IU, e.g., about 250 IU, about 500LU, about 750 IU, about 1000 IU, about 1500 IU, about 2000 IU, about3000 IU, about 4000 IU, about 5000 IU, or about 6000 IU.

In one embodiment, a pharmaceutical kit is provided which comprises afirst container comprising a lyophilized powder, where the powdercomprises (i) about 250 IU of a FVII polypeptide (e.g., a long-acting orshort-acting FVII polypeptide, e.g., rFVIIIFc), (ii) about 40 mg ofsucrose; (iii) about 36 mg of NaCl; (iv) about 3.1 mg of L-histidine;(v) about 2.4 mg of calcium chloride dihydrate; and (vi) about 0.40 mgof polysorbate 20 or polysorbate 80; and a second container comprisingsterilized water for injections at a volume sufficient to produce, whencombined with the lyophilized powder of the first container, a solutioncomprising: (i) about 83 IU/ml of a FVIII polypeptide (e.g., along-acting or short-acting FVII polypeptide, e.g., rFVIIIFc); (ii)about 1.33% (w/v) of sucrose; (iii) about 205 mM of NaCl; (iv) about6.64 mM L-histidine; (v) about 5.4 mM of calcium chloride; and (vi)about 0.013% (w/v) of polysorbate 20 or polysorbate 80.

In a further embodiment, a pharmaceutical kit is provided whichcomprises a first container comprising a lyophilized powder, where thepowder comprises: (i) about 500 IU of a FVII polypeptide (e.g., along-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc), (ii)about 40 mg of sucrose; (iii) about 36 mg of NaCl; (iv) about 3.1 mg ofL-histidine; (v) about 2.4 mg of calcium chloride dihydrate; and (vi)about 0.40 mg of polysorbate 20 or polysorbate 80; and a secondcontainer comprising sterilized water for injections at a volumesufficient to produce, when combined with the lyophilized powder of thefirst container, a solution comprising: (i) about 167 IU/ml of a FVIIIpolypeptide (e.g., a long-acting or short-acting FVIII polypeptide,e.g., rFVIIIFc); (ii) about 1.33% (w/v) of sucrose; (iii) about 205 mMof NaCl; (iv) about 6.64 mM L-histidine; (v) about 5.4 mM of calciumchloride; and (vi) about 0.013% (w/v) of polysorbate 20 or polysorbate80.

In a further embodiment, a pharmaceutical kit is provided whichcomprises a first container comprising a lyophilized powder, where thepowder comprises: (i) about 750 IU of a FVII polypeptide (e.g., along-acting or short-acting FVII polypeptide, e.g., rFVIIIFc), (ii)about 40 mg of sucrose; (iii) about 36 mg of NaCl; (iv) about 3.1 mg ofL-histidine; (v) about 2.4 mg of calcium chloride dihydrate; and (vi)about 0.40 mg of polysorbate 20 or polysorbate 80; and a secondcontainer comprising sterilized water for injections at a volumesufficient to produce, when combined with the lyophilized powder of thefirst container, a solution comprising: (i) about 250 IU/ml of a FVIIpolypeptide (e.g., a long-acting or short-acting FVIII polypeptide,e.g., rFVIIIFc); (ii) about 1.33% (w/v) of sucrose; (iii) about 205 mMof NaCl; (iv) about 6.64 mM L-histidine; (v) about 5.4 mM of calciumchloride; and (vi) about 0.013% (w/v) of polysorbate 20 or polysorbate80.

In a further embodiment, a pharmaceutical kit is provided whichcomprises a first container comprising a lyophilized powder, where thepowder comprises: (i) about 1000 IU of a FVIII polypeptide (e.g., along-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc), (ii)about 40 mg of sucrose; (iii) about 36 mg of NaCl; (iv) about 3.1 mg ofL-histidine; (v) about 2.4 mg of calcium chloride dihydrate; and (vi)about 0.40 mg of polysorbate 20 or polysorbate 80; and a secondcontainer comprising sterilized water for injections at a volumesufficient to produce, when combined with the lyophilized powder of thefirst container, a solution comprising: (i) about 333 IU/ml of a FVIIIpolypeptide (e.g., a long-acting or short-acting FVIII polypeptide,e.g., rFVIIIFc); (ii) about 1.33% (w/v) of sucrose; (iii) about 205 mMof NaCl; (iv) about 6.64 mM L-histidine; (v) about 5.4 mM of calciumchloride; and (vi) about 0.013% (w/v) of polysorbate 20 or polysorbate80.

In a further embodiment, a pharmaceutical kit is provided whichcomprises a first container comprising a lyophilized powder, where thepowder comprises: (i) about 1500 IU of a FVIII polypeptide (e.g., along-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc), (ii)about 40 mg of sucrose; (iii) about 36 mg of NaCl; (iv) about 3.1 mg ofL-histidine; (v) about 2.4 mg of calcium chloride dihydrate; and (vi)about 0.40 mg of polysorbate 20 or polysorbate 80; and a secondcontainer comprising sterilized water for injections at a volumesufficient to produce, when combined with the lyophilized powder of thefirst container, a solution comprising: (i) about 500 IU/ml of a FVIIIpolypeptide (e.g., a long-acting or short-acting FVIII polypeptide,e.g., rFVIIIFc); (ii) about 1.33% (w/v) of sucrose; (iii) about 205 mMof NaCl; (iv) about 6.64 mM L-histidine; (v) about 5.4 mM of calciumchloride; and (vi) about 0.013% (w/v) of polysorbate 20 or polysorbate80.

In a further embodiment, a pharmaceutical kit is provided whichcomprises a first container comprising a lyophilized powder, where thepowder comprises: (i) about 2000 IU of a FVIII polypeptide (e.g., along-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc), (ii)about 40 mg of sucrose; (iii) about 36 mg of NaCl; (iv) about 3.1 mg ofL-histidine; (v) about 2.4 mg of calcium chloride dihydrate; and (vi)about 0.40 mg of polysorbate 20 or polysorbate 80; and a secondcontainer comprising sterilized water for injections at a volumesufficient to produce, when combined with the lyophilized powder of thefirst container, a solution comprising: (i) about 667 IU/ml of a FVIIIpolypeptide (e.g., a long-acting or short-acting FVIII polypeptide,e.g., rFVIIIFc); (ii) about 1.33% (w/v) of sucrose; (iii) about 205 mMof NaCl; (iv) about 6.64 mM L-histidine; (v) about 5.4 mM of calciumchloride; and (vi) about 0.013% (w/v) of polysorbate 20 or polysorbate80.

In a further embodiment, a pharmaceutical kit is provided whichcomprises a first container comprising a lyophilized powder, where thepowder comprises: (i) about 3000 IU of a FVIII polypeptide (e.g., along-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc), (ii)about 40 mg of sucrose; (iii) about 36 mg of NaCl; (iv) about 3.1 mg ofL-histidine; (v) about 2.4 mg of calcium chloride dihydrate; and (vi)about 0.40 mg of polysorbate 20 or polysorbate 80; and a secondcontainer comprising sterilized water for injections at a volumesufficient to produce, when combined with the lyophilized powder of thefirst container, a solution comprising: (i) about 1000 IU/ml of a FVIIpolypeptide (e.g., a long-acting or short-acting FVIII polypeptide,e.g., rFVIIIFc); (ii) about 1.33% (w/v) of sucrose; (iii) about 205 mMof NaCl; (iv) about 6.64 mM L-histidine; (v) about 5.4 mM of calciumchloride; and (vi) about 0.013% (w/v) of polysorbate 20 or polysorbate80.

In a further embodiment, a pharmaceutical kit is provided whichcomprises a first container comprising a lyophilized powder, where thepowder comprises: (i) about 4000 IU of a FVII polypeptide (e.g., along-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc), (ii)about 40 mg of sucrose; (iii) about 36 mg of NaCl; (iv) about 3.1 mg ofL-histidine; (v) about 2.4 mg of calcium chloride dihydrate; and (vi)about 0.40 mg of polysorbate 20 or polysorbate 80; and a secondcontainer comprising sterilized water for injections at a volumesufficient to produce, when combined with the lyophilized powder of thefirst container, a solution comprising: (i) about 1333 IU/ml of a FVIIIpolypeptide (e.g., a long-acting or short-acting FVIII polypeptide,e.g., rFVIIIFc); (ii) about 1.33% (w/v) of sucrose; (iii) about 205 mMof NaCl; (iv) about 6.64 mM L-histidine; (v) about 5.4 mM of calciumchloride; and (vi) about 0.013% (w/v) of polysorbate 20 or polysorbate80.

In a further embodiment, a pharmaceutical kit is provided whichcomprises a first container comprising a lyophilized powder, where thepowder comprises: (i) about 5000 IU of a FVII polypeptide (e.g., along-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc), (ii)about 40 mg of sucrose; (iii) about 36 mg of NaCl; (iv) about 3.1 mg ofL-histidine; (v) about 2.4 mg of calcium chloride dihydrate; and (vi)about 0.40 mg of polysorbate 20 or polysorbate 80; and a secondcontainer comprising sterilized water for injections at a volumesufficient to produce, when combined with the lyophilized powder of thefirst container, a solution comprising: (i) about 1667 IU/ml of a FVIIIpolypeptide (e.g., a long-acting or short-acting FVIII polypeptide,e.g., rFVIIIFc); (ii) about 1.33% (w/v) of sucrose; (iii) about 205 mMof NaCl; (iv) about 6.64 mM L-histidine; (v) about 5.4 mM of calciumchloride; and (vi) about 0.013% (w/v) of polysorbate 20 or polysorbate80.

In a further embodiment, a pharmaceutical kit is provided whichcomprises a first container comprising a lyophilized powder, where thepowder comprises: (i) about 6000 IU of a FVIII polypeptide (e.g., along-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc), (ii)about 40 mg of sucrose; (iii) about 36 mg of NaCl; (iv) about 3.1 mg ofL-histidine; (v) about 2.4 mg of calcium chloride dihydrate; and (vi)about 0.40 mg of polysorbate 20 or polysorbate 80; and a secondcontainer comprising sterilized water for injections at a volumesufficient to produce, when combined with the lyophilized powder of thefirst container, a solution comprising: (i) about 2000 IU/ml of a FVIIIpolypeptide (e.g., a long-acting or short-acting FVIII polypeptide,e.g., rFVIIIFc); (ii) about 1.33% (w/v) of sucrose; (iii) about 205 mMof NaCl; (iv) about 6.64 mM L-histidine; (v) about 5.4 mM of calciumchloride; and (vi) about 0.013% (w/v) of polysorbate 20 or polysorbate80.

In a further embodiment, a pharmaceutical kit is provided whichcomprises a first container comprising a lyophilized powder, where thepowder comprises: (i) about 250 IU of a FVII polypeptide (e.g., along-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc), (ii)about 40 mg of sucrose; (iii) about 36 mg of NaCl; (iv) about 3.1 mg ofL-histidine; (v) about 2.4 mg of calcium chloride dihydrate; and (vi)about 0.40 mg of polysorbate 20 or polysorbate 80; and a secondcontainer comprising sterilized water for injections at a volumesufficient to produce, when combined with the lyophilized powder of thefirst container, a solution comprising: (i) about 83 IU/ml of a FVIIIpolypeptide (e.g., a long-acting or short-acting FVIII polypeptide,e.g., rFVIIIFc); (ii) about 13.3 mg/ml of sucrose; (iii) about 12.0mg/ml of NaCl; (iv) about 1.03 mg/ml L-histidine; (v) about 0.80 mg/mlof calcium chloride dihydrate; and (vi) about 0.13 mg/ml of polysorbate20 or polysorbate 80.

In a further embodiment, a pharmaceutical kit is provided whichcomprises a first container comprising a lyophilized powder, where thepowder comprises: (i) about 500 IU of a FVIII polypeptide (e.g., along-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc), (ii)about 40 mg of sucrose; (iii) about 36 mg of NaCl; (iv) about 3.1 mg ofL-histidine; (v) about 2.4 mg of calcium chloride dihydrate; and (vi)about 0.40 mg of polysorbate 20 or polysorbate 80; and a secondcontainer comprising sterilized water for injections at a volumesufficient to produce, when combined with the lyophilized powder of thefirst container, a solution comprising: (i) about 167 IU/ml of a FVIIIpolypeptide (e.g., a long-acting or short-acting FVIII polypeptide,e.g., rFVIIIFc); (ii) about 13.3 mg/ml of sucrose; (iii) about 12.0mg/ml of NaCl; (iv) about 1.03 mg/ml L-histidine; (v) about 0.80 mg/mlof calcium chloride dihydrate; and (vi) about 0.13 mg/ml of polysorbate20 or polysorbate 80.

In a further embodiment, a pharmaceutical kit is provided whichcomprises a first container comprising a lyophilized powder, where thepowder comprises: (i) about 750 IU of a FVIII polypeptide (e.g., along-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc), (ii)about 40 mg of sucrose; (iii) about 36 mg of NaCl; (iv) about 3.1 mg ofL-histidine; (v) about 2.4 mg of calcium chloride dihydrate; and (vi)about 0.40 mg of polysorbate 20 or polysorbate 80; and a secondcontainer comprising sterilized water for injections at a volumesufficient to produce, when combined with the lyophilized powder of thefirst container, a solution comprising: (i) about 250 IU/ml of a FVIIIpolypeptide (e.g., a long-acting or short-acting FVIII polypeptide,e.g., rFVIIIFc); (ii) about 13.3 mg/ml of sucrose; (iii) about 12.0mg/ml of NaCl; (iv) about 1.03 mg/ml L-histidine; (v) about 0.80 mg/mlof calcium chloride dihydrate; and (vi) about 0.13 mg/ml of polysorbate20 or polysorbate 80.

In a further embodiment, a pharmaceutical kit is provided whichcomprises a first container comprising a lyophilized powder, where thepowder comprises: (i) about 1000 IU of a FVIII polypeptide (e.g., along-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc), (ii)about 40 mg of sucrose; (iii) about 36 mg of NaCl; (iv) about 3.1 mg ofL-histidine; (v) about 2.4 mg of calcium chloride dihydrate; and (vi)about 0.40 mg of polysorbate 20 or polysorbate 80; and a secondcontainer comprising sterilized water for injections at a volumesufficient to produce, when combined with the lyophilized powder of thefirst container, a solution comprising: (i) about 333 IU/ml of a FVIIIpolypeptide (e.g., a long-acting or short-acting FVIII polypeptide,e.g., rFVIIIFc); (ii) about 13.3 mg/ml of sucrose; (iii) about 12.0mg/ml of NaCl; (iv) about 1.03 mg/ml L-histidine; (v) about 0.80 mg/mlof calcium chloride dihydrate; and (vi) about 0.13 mg/ml of polysorbate20 or polysorbate 80.

In a further embodiment, a pharmaceutical kit is provided whichcomprises a first container comprising a lyophilized powder, where thepowder comprises: (i) about 1500 IU of a FVIII polypeptide (e.g., along-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc), (ii)about 40 mg of sucrose; (iii) about 36 mg of NaCl; (iv) about 3.1 mg ofL-histidine; (v) about 2.4 mg of calcium chloride dihydrate; and (vi)about 0.40 mg of polysorbate 20 or polysorbate 80; and a secondcontainer comprising sterilized water for injections at a volumesufficient to produce, when combined with the lyophilized powder of thefirst container, a solution comprising: (i) about 500 IU/ml of a FVIIIpolypeptide (e.g., a long-acting or short-acting FVIII polypeptide,e.g., rFVIIIFc); (ii) about 13.3 mg/ml of sucrose; (iii) about 12.0mg/ml of NaCl; (iv) about 1.03 mg/ml L-histidine; (v) about 0.80 mg/mlof calcium chloride dihydrate; and (vi) about 0.13 mg/ml of polysorbate20 or polysorbate 80.

In a further embodiment, a pharmaceutical kit is provided whichcomprises a first container comprising a lyophilized powder, where thepowder comprises: (i) about 2000 IU of a FVIII polypeptide (e.g., along-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc), (ii)about 40 mg of sucrose; (iii) about 36 mg of NaCl; (iv) about 3.1 mg ofL-histidine; (v) about 2.4 mg of calcium chloride dihydrate; and (vi)about 0.40 mg of polysorbate 20 or polysorbate 80; and a secondcontainer comprising sterilized water for injections at a volumesufficient to produce, when combined with the lyophilized powder of thefirst container, a solution comprising: (i) about 667 IU/ml of a FVIIpolypeptide (e.g., a long-acting or short-acting FVII polypeptide, e.g.,rFVIIIFc); (ii) about 13.3 mg/ml of sucrose; (iii) about 12.0 mg/ml ofNaCl; (iv) about 1.03 mg/ml L-histidine; (v) about 0.80 mg/ml of calciumchloride dihydrate; and (vi) about 0.13 mg/ml of polysorbate 20 orpolysorbate 80.

In a further embodiment, a pharmaceutical kit is provided whichcomprises a first container comprising a lyophilized powder, where thepowder comprises: (i) about 3000 IU of a FVII polypeptide (e.g., along-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc), (ii)about 40 mg of sucrose; (iii) about 36 mg of NaCl; (iv) about 3.1 mg ofL-histidine; (v) about 2.4 mg of calcium chloride dihydrate; and (vi)about 0.40 mg of polysorbate 20 or polysorbate 80; and a secondcontainer comprising sterilized water for injections at a volumesufficient to produce, when combined with the lyophilized powder of thefirst container, a solution comprising: (i) about 1000 IU/ml of a FVIIIpolypeptide (e.g., a long-acting or short-acting FVIII polypeptide,e.g., rFVIIIFc); (ii) about 13.3 mg/ml of sucrose; (iii) about 12.0mg/ml of NaCl; (iv) about 1.03 mg/ml L-histidine; (v) about 0.80 mg/mlof calcium chloride dihydrate; and (vi) about 0.13 mg/ml of polysorbate20 or polysorbate 80.

In a further embodiment, a pharmaceutical kit is provided whichcomprises a first container comprising a lyophilized powder, where thepowder comprises: (i) about 4000 IU of a FVII polypeptide (e.g., along-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc), (ii)about 40 mg of sucrose; (iii) about 36 mg of NaCl; (iv) about 3.1 mg ofL-histidine; (v) about 2.4 mg of calcium chloride dihydrate; and (vi)about 0.40 mg of polysorbate 20 or polysorbate 80; and a secondcontainer comprising sterilized water for injections at a volumesufficient to produce, when combined with the lyophilized powder of thefirst container, a solution comprising: (i) about 1333 IU/ml of a FVIIpolypeptide (e.g., a long-acting or short-acting FVIII polypeptide,e.g., rFVIIIFc); (ii) about 13.3 mg/ml of sucrose; (iii) about 12.0mg/ml of NaCl; (iv) about 1.03 mg/ml L-histidine; (v) about 0.80 mg/mlof calcium chloride dihydrate; and (vi) about 0.13 mg/ml of polysorbate20 or polysorbate 80.

In a further embodiment, a pharmaceutical kit is provided whichcomprises a first container comprising a lyophilized powder, where thepowder comprises: (i) about 5000 IU of a FVIII polypeptide (e.g., along-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc), (ii)about 40 mg of sucrose; (iii) about 36 mg of NaCl; (iv) about 3.1 mg ofL-histidine; (v) about 2.4 mg of calcium chloride dihydrate; and (vi)about 0.40 mg of polysorbate 20 or polysorbate 80; and a secondcontainer comprising sterilized water for injections at a volumesufficient to produce, when combined with the lyophilized powder of thefirst container, a solution comprising: (i) about 1667 IU/ml of a FVIIIpolypeptide (e.g., a long-acting or short-acting FVIII polypeptide,e.g., rFVIIIFc); (ii) about 13.3 mg/ml of sucrose; (iii) about 12.0mg/ml of NaCl; (iv) about 1.03 mg/ml L-histidine; (v) about 0.80 mg/mlof calcium chloride dihydrate; and (vi) about 0.13 mg/ml of polysorbate20 or polysorbate 80.

In a further embodiment, a pharmaceutical kit is provided whichcomprises a first container comprising a lyophilized powder, where thepowder comprises: (i) about 6000 IU of a FVII polypeptide (e.g., along-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc), (ii)about 40 mg of sucrose; (iii) about 36 mg of NaCl; (iv) about 3.1 mg ofL-histidine; (v) about 2.4 mg of calcium chloride dihydrate; and (vi)about 0.40 mg of polysorbate 20 or polysorbate 80; and a secondcontainer comprising sterilized water for injections at a volumesufficient to produce, when combined with the lyophilized powder of thefirst container, a solution comprising: (i) about 2000 IU/ml of a FVIIIpolypeptide (e.g., a long-acting or short-acting FVIII polypeptide,e.g., rFVIIIFc); (ii) about 13.3 mg/ml of sucrose; (iii) about 12.0mg/ml of NaCl; (iv) about 1.03 mg/ml L-histidine; (v) about 0.80 mg/mlof calcium chloride dihydrate; and (vi) about 0.13 mg/ml of polysorbate20 or polysorbate 80.

In certain embodiments the first container of a pharmaceutical kitprovided herein is a glass vial comprising a rubber stopper. In certainembodiments, the second container a pharmaceutical kit provided hereinis a syringe body, associated with a plunger. In certain embodiments,the syringe is a pre-filled syringe containing the diluent. In certainembodiments, a pharmaceutical kit provided herein further comprises anadaptor to connect the glass vial to the syringe body. In certainembodiments a pharmaceutical kit provided herein further comprisesinfusion tubing associated with a needle to be connected to the syringe,suitable for intravenous infusion.

In certain embodiments, a desired dose of a FVIII polypeptide (e.g., along-acting or short-acting FVIII polypeptide, e.g., rFVIIIFc) can beachieved through the use of one pharmaceutical kit as provided herein.In certain embodiments, more than one pharmaceutical kit can be used toachieve a desired dose. Provided herein is a method of combining, orpooling the formulations contained in two or more pharmaceutical kits asprovided herein in order to achieve a desired dose.

In some embodiments, the FVIII polypeptide for the pharmaceuticalcomposition is a short-acting FVIII polypeptide. In other embodiments,the FVIII polypeptide for the pharmaceutical composition is along-acting FVIII polypeptide.

In some embodiments, a long-acting FVIII polypeptide in a pharmaceuticalcomposition comprises a Factor VIII portion and a non-Factor VIIIportion, e.g., a heterologous moiety. In one embodiment, theheterologous moiety is capable of extending in vivo or in vitrohalf-life of the FVII polypeptide. Exemplary non-Factor VII portionsinclude, but are not limited to, Fc, albumin, a PAS sequence,transferrin, CTP (28 amino acid C-terminal peptide (CTP) of humanchorionic gonadotropin (hCG) with its 4 O-glycans), polyethylene glycol(PEG), hydroxyethyl starch (HES), albumin binding polypeptide,albumin-binding small molecules, or any combination thereof. Exemplarylong-acting polypeptides of the invention include, e.g., Factor VIII-Fcpolypeptides, Factor VII-albumin polypeptides, Factor VII-PASpolypeptides, Factor VIII-transferrin polypeptides, Factor VIII-CTPpolypeptides, Factor VIII-PEG polypeptides, Factor VHI-HES polypeptides,Factor VIII-albumin binding polypeptide polypeptides, or FactorVIII-albumin-binding small molecule polypeptides.

The Factor VIII (the Factor VII portion of a long-acting FVIIIpolypeptide or FVIII of a short-acting FVIII polypeptide) can be atleast 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical toa Factor VIII amino acid sequence shown in Table 11 without a signalsequence (amino acids 20 to 1457 of SEQ ID NO:2; and amino acids 20 to2351 of SEQ ID NO:6), wherein the Factor VIII portion has Factor VIIIactivity. The Factor VIII (the Factor VIII portion of a chimericpolypeptide) can be identical to a Factor VIII amino acid sequence shownin Table 11 without a signal sequence (amino acids 20 to 1457 of SEQ IDNO:2; and amino acids 20 to 2351 of SEQ ID NO:6).

The Factor VIII (the Factor VIII portion of a long-acting FVIIIpolypeptide or FVIII of a short-acting FVIII polypeptide) can be atleast 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical toa Factor VII amino acid sequence shown in Table 11 with a signalsequence (amino acids 1 to 1457 of SEQ ID NO:2 and amino acids 1 to 2351of SEQ ID NO:6), wherein the Factor VII portion has Factor VI activity.The Factor VIII (the Factor VII portion of a long-acting FVIIpolypeptide or a short-acting FVIII polypeptide) can be identical to aFactor VIII amino acid sequence shown in Table 11 with a signal sequence(amino acids 1 to 1457 of SEQ ID NO:2 and amino acids 1 to 2351 of SEQID NO:6).

The long-acting polypeptide can comprise a sequence at least 60%, 70%,80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the Factor VIIand Fc amino acid sequence shown in Table 11A(i) without a signalsequence (amino acids 20 to 1684 of SEQ ID NO:2) or at least 60%, 70%,80%, 90%0, 95%, 96%, 97%, 98%, 99%, or 100% identical to the Factor VIIIand Fc amino acid sequence shown in Table 11A(i) with a signal sequence(amino acids 1 to 1684 of SEQ ID NO:2), wherein the sequence has FactorVIII activity. The Factor VIII activity can be measured by activatedPartial Thromboplastin Time (aPTT) assay, chromogenic assay, or otherknown methods. The long-acting FVIII polypeptide can comprise thesequence identical to the Factor VIII and Fc amino acid sequence shownin Table 11A(i) without a signal sequence (amino acids 20 to 1684 of SEQID NO:2) or identical to the Factor VIII and Fc amino acid sequenceshown in Table 11A(i) with a signal sequence (amino acids 1 to 1684 ofSEQ ID NO:2).

As discussed above, exemplary long-acting polypeptides also includeFactor VIII fused to one or more albumin polypeptides, albumin bindingpolypeptides, or albumin-binding small molecules. In one embodiment, thealbumin is human albumin. The albumin or albumin binding protein can befused to either the N-terminal end of FVII or to the C-terminal end ofFVIII or inserted between two amino acids in FVIII. Examples of albumin,e.g., fragments thereof, that can be used in the present invention areknown. e.g., U.S. Pat. Nos. 7,592,010; 6,686,179; and Schulte,Thrombosis Res. 124 Suppl. 2:S6-S8 (2009), each of which is incorporatedherein by reference in its entirety.

The albumin binding polypeptides can compromise, without limitation,bacterial albumin-binding domains, albumin-binding peptides, oralbumin-binding antibody fragments that can bind to albumin. Domain 3from streptococcal protein G, as disclosed by Kraulis et al., FEBS Lett.378:190-194 (1996) and Linhult et al., Protein Sci. 11:206-213 (2002) isan example of a bacterial albumin-binding domain. Examples ofalbumin-binding peptides include a series of peptides having the coresequence DICLPRWGCLW (SEQ ID NO: 7). See, e.g., Dennis et al., J. Biol.Chem. 2002, 277: 35035-35043 (2002). Examples of albumin-bindingantibody fragments are disclosed in Muller and Kontermann, Curr. Opin.Mol. Ther. 9:319-326 (2007); Rooverset et al., Cancer Immunol.Immunother. 56:303-317 (2007), and Holt et al., Prot. Eng. Design Sci.,21:283-288 (2008), which are incorporated herein by reference in theirentireties.

In certain aspects, a long-acting FVII polypeptide of the inventioncomprises at least one attachment site for a non-polypeptide smallmolecule, variant, or derivative that can bind to albumin thereof. Anexample of such albumin binding moieties is2-(3-maleimidopropanamido)-6-(4-(4-iodophenyl)butanamido)hexanoate(“Albu” tag) as disclosed by Trusselet et al., Bioconjugate Chem.20:2286-2292 (2009).

As discussed above, exemplary long-acting polypeptides also includeFactor VIII fused to at least one C-terminal peptide (CTP) of the 0subunit of human chorionic gonadotropin or fragment, variant, orderivative thereof. The CTP can be fused to Factor VIII either theN-terminal end of FVIII or to the C-terminal end of FVIII or insertedbetween two amino acids in FVIII. One or more CTP peptides fused to orinserted into a recombinant protein is known to increase the in vivohalf-life of that protein. See, e.g., U.S. Pat. No. 5,712,122,incorporated by reference herein in its entirety. Exemplary CTP peptidesinclude DPRFQDSSSSKAPPPSLPSPSRLPGPSDTPIL (SEQ ID NO: 8) orSSSSKAPPPSLPSPSRLPGPSDTPILPQ. (SEQ ID NO: 9). See, e.g., U.S. PatentApplication Publication No. US 2009/0087411 A1, incorporated byreference.

As discussed above, exemplary long-acting FVIII polypeptides alsoinclude Factor VIII fused to at least one PAS sequence or fragment,variant, or derivative thereof. The PAS sequence can be fused to eitherthe N-terminal end of FVIII or to the C-terminal end of FVIII orinserted between two amino acids in FVIII. A PAS peptide or PASsequence, as used herein, means an amino acid sequence comprising mainlyalanine and serine residues or comprising mainly alanine, serine, andproline residues, the amino acid sequence forming random coilconformation under physiological conditions. Accordingly, the PASsequence is a building block, an amino acid polymer, or a sequencecassette comprising, consisting essentially of, or consisting ofalanine, serine, and proline which can be used as a part of theheterologous moiety in the chimeric protein. An amino acid polymer alsocan form random coil conformation when residues other than alanine,serine, and proline are added as a minor constituent in the PASsequence. By “minor constituent” is meant that that amino acids otherthan alanine, serine, and proline can be added in the PAS sequence to acertain degree, e.g., up to about 12%, i.e., about 12 of 100 amino acidsof the PAS sequence, up to about 10%, up to about 9%, up to about 8%,about 6%, about 5%, about 4%, about 3%, i.e. about 2%, or about 1%, ofthe amino acids. The amino acids different from alanine, serine andproline can be selected from the group consisting of Arg, Asn, Asp, Cys,GIn, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, Tyr, and Val.Under physiological conditions, a PAS peptide forms a random coilconformation and thereby can mediate an increased in vivo and/or invitro stability to a recombinant protein of the invention, and hasprocoagulant activity.

Non-limiting examples of the PAS peptides include ASPAAPAPASPAAPAPSAPA(SEQ ID NO: 10), AAPASPAPAAPSAPAPAAPS (SEQ ID NO: 11),APSSPSPSAPSSPSPASPSS (SEQ ID NO: 12), APSSPSPSAPSSPSPASPS (SEQ ID NO:13), SSPSAPSPSSPASPSPSSPA (SEQ ID NO: 14), AASPAAPSAPPAAASPAAPSAPPA (SEQID NO: 15), ASAAAPAAASAAASAPSAAA (SEQ ID NO: 16) or any variants,derivatives, fragments, or combinations thereof. Additional examples ofPAS sequences are known from, e.g., US Pat. Publ. No. 2010/0292130 A1and PCT Appl. Publ. No. WO 2008/155134 A1. European issued patentEP2173890.

As discussed above, exemplary long-acting FVIII polypeptides alsoinclude Factor VIII fused to at least one transferrin peptide orfragment, variant, or derivative thereof. At least one transferrinpeptide can be fused to either the N-terminal end of FVIII or to theC-terminal end of FVIII or inserted between two amino acids in FVIII.Any transferrin can be fused to or inserted into a recombinant FVIIIprotein of the invention. As an example, wild-type human Tf (Tf) is a679 amino acid protein, of approximately 75 KDa (not accounting forglycosylation), with two main domains, N (about 330 amino acids) and C(about 340 amino acids), which appear to originate from a geneduplication. See GenBank accession numbers NM001063, XM002793, M12530,XM039845, XM 039847 and S95936 (www.ncbi.nlm.nih.gov), all of which areherein incorporated by reference in their entirety.

Transferrin transports iron through transferrin receptor (TfR)-mediatedendocytosis. After the iron is released into an endosomal compartmentand Tf-TfR complex is recycled to cell surface, the Tf is released backextracellular space for next cycle of iron transporting. Tf possesses along half-life that is in excess of 14-17 days (Li et al., TrendsPharmacol. Sci. 23:206-209 (2002)). Transferrin fusion proteins havebeen studied for half-life extension, targeted deliver for cancertherapies, oral delivery and sustained activation of proinsulin(Brandsma et al., Biotechnol. Adv., 29: 230-238 (2011); Bai et al.,Proc. Natl. Acad. Sci. USA 102:7292-7296 (2005); Kim et al., J.Pharmacol. Erp. Ther., 334:682-692 (2010); Wang et al., J. ControlledRelease 155:386-392 (2011)).

As discussed above, exemplary long-acting FVIII polypeptides alsoinclude Factor VIII fused to at least one polyethylene glycol (PEG)moieties.

PEGylated FVIII can refer to a conjugate formed between FVIII and atleast one polyethylene glycol (PEG) molecule. PEG is commerciallyavailable in a large variety of molecular weights and average molecularweight ranges. Typical examples of PEG average molecular weight rangesinclude, but are not limited to, about 200, about 300, about 400, about600, about 1000, about 1300-1600, about 1450, about 2000, about 3000,about 3000-3750, about 3350, about 3000-7000, about 3500-4500, about5000-7000, about 7000-9000, about 8000, about 10000, about 8500-11500,about 16000-24000, about 35000, about 40000, about 60000, and about80000 daltons. These average molecular weights are provided merely asexamples and are not meant to be limiting in any way.

A long-acting FVIII polypeptide useful for the invention can bePEGylated to include mono- or poly-(e.g., 2-4) PEG moieties. PEGylationcan be carried out by any of the PEGylation reactions known in the art.Methods for preparing a PEGylated protein product will generally include(i) reacting a polypeptide with polyethylene glycol (such as a reactiveester or aldehyde derivative of PEG) under conditions whereby thepeptide of the invention becomes attached to one or more PEG groups; and(ii) obtaining the reaction product(s). In general, the optimal reactionconditions for the reactions will be determined case by case based onknown parameters and the desired result.

There are a number of PEG attachment methods available to those skilledin the art, for example Malik F et al., Exp. Hemaol. 20:1028-35 (1992);Francis, Focus on Growth Factors 3(2):4-10 (1992); European Pat. Pub.Nos. EP0401384, EP0154316, and EP0401384; and International Pat. Appl.Pub. Nos. WO92/16221 and W95/34326. As a non-limiting example, FVIIIvariants can contain cysteine substitutions in one or more insertionsites in FVIII, and the cysteines can be further conjugated to PEGpolymer. See Mei et al., Blood 116:270-279 (2010) and U.S. Pat. No.7,632,921, which are incorporated herein by reference in theirentireties.

As discussed above, exemplary long-acting FVIII polypeptides alsoinclude Factor VIII fused to at least one hydroxyethyl starch (HES)polymer. HES is a derivative of naturally occurring amylopectin and isdegraded by alpha-amylase in the body. HES exhibits advantageousbiological properties and is used as a blood volume replacement agentand in hemodilution therapy in the clinics. See, e.g., Sommermeyer etal., Krankenhauspharmazie 8:271-278 (1987); and Weidler et al.,Arzneim.-ForschungDrug Res. 41: 494-498 (1991).

HES is mainly characterized by the molecular weight distribution and thedegree of substitution. HES has a mean molecular weight (weight mean) offrom 1 to 300 kD, from 2 to 200 kD, from 3 to 100 kD, or from 4 to 70kD. Hydroxyethyl starch can further exhibit a molar degree ofsubstitution of from 0.1 to 3, from 0.1 to 2, from 0.1 to 0.9, or from0.1 to 0.8, and a ratio between C2:C6 substitution in the range of from2 to 20 with respect to the hydroxyethyl groups. HES with a meanmolecular weight of about 130 kD is VOLUVEN® from Fresenius. VOLUVEN® isan artificial colloid, employed, e.g., for volume replacement used inthe therapeutic indication for therapy and prophylaxis of hypovolaemia.There are a number of HES attachment methods available to those skilledin the art, e.g., the same PEG attachment methods described above.

In some embodiments, a long-acting FVIII polypeptide comprising a FVIIportion has an increased half-life (tin) over a polypeptide consistingof the same FVII portion without the non-FVIII portion. A long-actingFVIII polypeptide with an increased tin can be referred to herein as along-lasting FVIII. Long-acting chimeric Factor VIII polypeptidesinclude, e.g., Factor VIII fused to Fc (including, e.g., chimeric FactorVIII polypeptides in the form of a hybrid such as a FVIIIFc monomerdimer hybrid; see Example, Table 11A; and U.S. Pat. Nos. 7,404,956 and7,348,004), and Factor VIII fused to albumin.

The Factor VIII polypeptide as used herein is functional factor VIIpolypeptide in its normal role in coagulation, unless otherwisespecified. Thus, the term Factor VII includes variant polypeptides thatare functional. Factor VIII proteins can be the human, porcine, canine,and murine factor VIII proteins. The full length polypeptide andpolynucleotide sequences are known, as are many functional fragments,mutants and modified versions. Examples of human factor VIII sequencesare shown as subsequences in SEQ ID NOs: 2 or 6 (Table 11). Factor VIIIpolypeptides include, e.g., full-length factor VIII, full-length factorVII minus Met at the N-terminus, mature factor VII (minus the signalsequence), mature factor VIII with an additional Met at the N-terminus,and/or factor VII with a full or partial deletion of the B domain.Factor VII variants include B domain deletions, whether partial or fulldeletions.

A great many functional factor VIII variants are known, as is discussedabove and below. In addition, hundreds of nonfunctional mutations infactor VIII have been identified in hemophilia patients, and it has beendetermined that the effect of these mutations on factor VIII function isdue more to where they lie within the 3-dimensional structure of factorVIII than on the nature of the substitution (Cutler et al., Hum. Mutat.19:274-8 (2002)), incorporated herein by reference in its entirety. Inaddition, comparisons between factor VIII from humans and other specieshave identified conserved residues that are likely to be required forfunction (Cameron et al., Thromb. Haemost. 79:317-22 (1998); U.S. Pat.No. 6,251,632), incorporated herein by reference in its entirety.

The human factor VIII gene was isolated and expressed in mammalian cells(Toole, J. J., et al., Nature 312:342-347 (1984); Gitschier, J., et al.,Nature 312:326-330 (1984); Wood, W. I., et al., Nature 312:330-337(1984); Vehar, G. A., et al., Nature 312:337-342 (1984); WO 87/04187; WO88/08035; WO 88/03558; U.S. Pat. No. 4,757,006), each of which isincorporated herein by reference in its entirety, and the amino acidsequence was deduced from cDNA. Capon et al., U.S. Pat. No. 4,965,199,incorporated herein by reference in its entirety, discloses arecombinant DNA method for producing factor VIII in mammalian host cellsand purification of human factor VIII. Human factor VIII expression inCHO (Chinese hamster ovary) cells and BHK (baby hamster kidney cells)has been reported. Human factor VIII has been modified to delete part orall of the B domain (U.S. Pat. Nos. 4,994,371 and 4,868,112, each ofwhich is incorporated herein by reference in its entirety), andreplacement of the human factor VIII B domain with the human factor V Bdomain has been performed (U.S. Pat. No. 5,004,803, incorporated hereinby reference in its entirety). The cDNA sequence encoding human factorVIII and predicted amino acid sequence are shown in SEQ ID NOs:1 and 2,respectively, of US Application Publ. No. 2005/0100990, incorporatedherein by reference in its entirety.

U.S. Pat. No. 5,859,204, Lollar, J. S., incorporated herein by referencein its entirety, reports functional mutants of factor VIII havingreduced antigenicity and reduced immunoreactivity. U.S. Pat. No.6,376,463, Lollar, J. S., incorporated herein by reference in itsentirety, also reports mutants of factor VIII having reducedimmunoreactivity. US Application Publ. No. 2005/0100990, Saenko et al.,incorporated herein by reference in its entirety, reports functionalmutations in the A2 domain of factor VIII.

A number of functional factor VIII molecules, including B-domaindeletions, are disclosed in the following patents U.S. Pat. Nos.6,316,226 and 6,346,513, both assigned to Baxter; U.S. Pat. No.7,041,635 assigned to In2Gen; U.S. Pat. Nos. 5,789,203, 6,060,447,5,595,886, and 6,228,620 assigned to Chiron; U.S. Pat. Nos. 5,972,885and 6,048,720 assigned to Biovitrum, U.S. Pat. Nos. 5,543,502 and5,610,278 assigned to Novo Nordisk; U.S. Pat. No. 5,171,844 assigned toImmuno Ag; U.S. Pat. No. 5,112,950 assigned to Transgene S.A.; U.S. Pat.No. 4,868,112 assigned to Genetics Institute, each of which isincorporated herein by reference in its entirety.

The porcine factor VIII sequence is published, (Toole, J. J., et al.,Proc. Natl. Acad. Sci. USA 83:5939-5942 (1986)), incorporated herein byreference in its entirety, and the complete porcine cDNA sequenceobtained from PCR amplification of factor VIII sequences from a pigspleen cDNA library has been reported (Healey, J. F. et al., Blood88:4209-4214 (1996), incorporated herein by reference in its entirety).Hybrid human/porcine factor VIII having substitutions of all domains,all subunits, and specific amino acid sequences were disclosed in U.S.Pat. No. 5,364,771 by Lollar and Runge, and in WO 93/20093, incorporatedherein by reference in its entirety. More recently, the nucleotide andcorresponding amino acid sequences of the A1 and A2 domains of porcinefactor VIII and a chimeric factor VIII with porcine A1 and/or A2 domainssubstituted for the corresponding human domains were reported in WO94/11503, incorporated herein by reference in its entirety. U.S. Pat.No. 5,859,204, Lollar, J. S., also discloses the porcine cDNA anddeduced amino acid sequences. U.S. Pat. No. 6,458,563, incorporatedherein by reference in its entirety assigned to Emory discloses aB-domain deleted porcine Factor VIII.

A “B domain” of Factor VIII, as used herein, is the same as the B domainknown in the art that is defined by internal amino acid sequenceidentity and sites of proteolytic cleavage by thrombin, e.g., residuesSer741-Arg1648 of full length mature human factor VIII. The other humanfactor VIII domains are defined by the following amino acid residues:A1, residues Alal-Arg372; A2, residues Ser373-Arg740; A3, residuesSer1690-Ile2032; C1, residues Arg2033-Asn2172; C2, residuesSer2173-Tyr2332. The A3-C1-C2 sequence includes residuesSer1690-Tyr2332. The remaining sequence, residues Glu1649-Arg1689, isusually referred to as the factor VII light chain activation peptide.The locations of the boundaries for all of the domains, including the Bdomains, for porcine, mouse and canine factor VIII are also known in theart. In one embodiment, the B domain of Factor VIII is deleted (“Bdomain deleted factor VIII” or “BDD FVIII”). An example of a BDD FVIIIis REFACTO® (recombinant BDD FVIII), which has the sequence as theFactor VIII portion of the sequence in Table 1A(i) (amino acids 1 to1457 or 20 to 1457 of SEQ ID NO:2). In another embodiment, the B domaindeleted Factor VIII contains an intact intracellular processing site,which corresponds to Arginine at residue 754 of B domain deleted FactorVIII, which corresponds to Arginine residue 773 of SEQ ID NO: 2, orresidue 1648 of full-length Factor VIII, which corresponds to Arginineresidue 1667 of SEQ ID NO: 6. The sequence residue numbers used hereinwithout referring to any SEQ ID Numbers correspond to the Factor VIIsequence without the signal peptide sequence (19 amino acids) unlessotherwise indicated. For example, S743/Q1638 of full-length Factor VIIIcorresponds to S762/Q1657 of SEQ ID NO: 6 due to the 19 amino acidsignal peptide sequence. In other embodiments, the B domain deleted FVIIcomprises a substitution or mutation at an amino acid positioncorresponding to Arginine 1645, a substitution or mutation at an aminoacid position corresponding to Arginine 1648, or a substitution ormutation at amino acid positions corresponding to Arginine 1645 andArginine 1648 in full-length Factor VIII. In some embodiments, the aminoacid substituted at the amino acid position corresponding to Arginine1645 is a different amino acid from the amino acid substituted at theamino acid position corresponding to Arginine 1648. In certainembodiments, the substitution or mutation is an amino acid other thanarginine, e.g., alanine.

A “B domain deleted factor VIII” can have the full or partial deletionsdisclosed in U.S. Pat. Nos. 6,316,226, 6,346,513, 7,041,635, 5,789,203,6,060,447, 5,595,886, 6,228,620, 5,972,885, 6,048,720, 5,543,502,5,610,278, 5,171,844, 5,112,950, 4,868,112, and 6,458,563, each of whichis incorporated herein by reference in its entirety. In someembodiments, a B domain deleted factor VIII sequence of the presentinvention comprises any one of the deletions disclosed at col. 4, line 4to col. 5, line 28 and examples 1-5 of U.S. Pat. No. 6,316,226 (also inU.S. Pat. No. 6,346,513). In some embodiments, a B domain deleted factorVII of the present invention has a deletion disclosed at col. 2, lines26-51 and examples 5-8 of U.S. Pat. No. 5,789,203 (also U.S. Pat. Nos.6,060,447, 5,595,886, and 6,228,620). In some embodiments, a B domaindeleted factor VIII has a deletion described in col. 1, lines 25 to col.2, line 40 of U.S. Pat. No. 5,972,885; col. 6, lines 1-22 and example 1of U.S. Pat. No. 6,048,720; col. 2, lines 17-46 of U.S. Pat. No.5,543,502; col. 4, line 22 to col. 5, line 36 of U.S. Pat. No.5,171,844; col. 2, lines 55-68, FIG. 2, and example 1 of U.S. Pat. No.5,112,950; col. 2, line 2 to col. 19, line 21 and Table 2 of U.S. Pat.No. 4,868,112; col. 2, line 1 to col. 3, line 19, col. 3, line 40 tocol. 4, line 67, col. 7, line 43 to col. 8, line 26, and col. 11, line 5to col. 13, line 39 of U.S. Pat. No. 7,041,635; or col. 4, lines 25-53,of U.S. Pat. No. 6,458,563. In some embodiments, a B domain deletedfactor VIII has a deletion of most of the B domain, but still containsamino-terminal sequences of the B domain that are essential for in vivoproteolytic processing of the primary translation product into twopolypeptide chain (i.e., intracellular processing site), as disclosed inWO 91/09122, which is incorporated herein by reference in its entirety.In some embodiments, a B domain deleted factor VIII is constructed witha deletion of amino acids 747-1638, i.e., virtually a complete deletionof the B domain. Hoeben R. C., et al. J. Biol. Chem. 265 (13): 7318-7323(1990), incorporated herein by reference in its entirety. A B domaindeleted factor VIII can also contain a deletion of amino acids 771-1666or amino acids 868-1562 of factor VIII. Meulien P., et al. Protein Eng.2(4): 301-6 (1988), incorporated herein by reference in its entirety.Additional B domain deletions that are part of the invention include,e.g., deletion of amino acids 982 through 1562 or 760 through 1639(Toole et al., Proc. Natl. Acad Sci. U.S.A. 83:5939-5942 (1986)), 797through 1562 (Eaton et al., Biochemistry 25:8343-8347 (1986)), 741through 1646 (Kaufman (PCT published application No. WO 87/04187)),747-1560 (Sarver et al., DNA 6:553-564 (1987)), 741 through 1648 (Pasek(PCT application No. 88/00831)), 816 through 1598 or 741 through 1689(Lagner (Behring Inst. Mitt. (1988) No 82:16-25, EP 295597)), each ofwhich is incorporated herein by reference in its entirety. In someembodiments, B domain deleted FVIII comprises a partial deletion in Bdomain, i.e., having 21 amino acids from B domain (i.e.,SFSQNSRHPSQNPPVLKRHQR, which is SEQ ID NO: 17) disclosed in USPublication No. 20100286067 and US Publication No. US 20120093840, bothof which are incorporated herein by reference in their entireties. Eachof the foregoing deletions can be made in any Factor VIII sequence.

In one embodiment, the B domain deleted Factor VIII portion in a FVIIIpolypeptide is processed into two chains connected (or associated) by ametal bond, the first chain comprising a heavy chain (A1-A2-partial B)and a second chain comprising a light chain (A3-C1-C2). In anotherembodiment, the B domain deleted Factor VIII portion is a single chainFactor VIII. The single chain Factor VIII can comprise an intracellularprocessing site, which corresponds to Arginine at residue 754 of Bdomain deleted Factor VIII (residue 773 of SEQ ID NO: 2) or at residue1648 of full-length Factor VIII (residue 1657 of SEQ ID NO: 6).

The metal bond between the heavy chain and the light chain can be anymetal known in the art. For example, the metals useful for the inventioncan be a divalent metal ion. The metals that can be used to associatethe heavy chain and light chain include, but not limited to, Ca²⁺, Mn²⁺,or Cu. Fatouros et al., Intern. J. Pharm. 155(1): 121-131 (1997);Wakabayashi et al., JBC. 279(13): 12677-12684 (2004).

A FVIII polypeptide used herein can comprise processed Factor VIII orsingle chain Factor VIII or a combination thereof. “Processed FactorVIII,” as used herein means Factor VIII that has been cleaved atArginine 1648 (for full-length Factor VIII) or Arginine 754 (forB-domain deleted Factor VIII), i.e., intracellular processing site. Dueto the cleavage at the intracellular processing site, processed FactorVIII comprises two polypeptide chains, the first chain being a heavychain and the second chain being a light chain. For example, theprocessed Factor VIII-Fc fusion protein (i.e., Heavy chain and Lightchain fused to Fc) run at approximately 90 kDa and 130 kDa on anon-reducing SDS-PAGE, respectively, and 90 kDa and 105 kDa on areducing SDS-PAGE, respectively. Therefore, in one embodiment, at leastabout 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about100% of the Factor VIII portion in the long-acting polypeptide or in theshort-acting FVIII polypeptide is processed Factor VIII. In anotherembodiment, about 50%, about 60%, about 70%, about 75%, about 80%, about85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%,or about 100% of the Factor VIII portion in the long-acting polypeptideor in the short-acting FVIII polypeptide is processed Factor VIII. In aparticular embodiment, the FVIII polypeptide comprising processed FactorVIII is purified (or isolated) from the polypeptide comprising singlechain Factor VIII, and at least about 75%, about 80%, about 85%, about90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about100% of the Factor VIII portion in the long-acting polypeptide or in theshort-acting FVIII polypeptide is processed Factor VIII. In someembodiments, the FVIII polypeptide comprises about 15% to 25% of singlechain FVII polypeptide and about 75% to about 85% of processed FVIIIpolypeptide.

“Single chain Factor VIII,” “SC Factor VIII,” or “SCFVIII” as usedherein means Factor VIII that has not been cleaved at the Arginine site(residue 1648 for full-length Factor VIII (i.e., residue 1667 of SEQ IDNO: 6) or residue 754 for B-domain deleted Factor VIII (i.e., residue773 of SEQ ID NO: 2). Therefore, single chain Factor VIII in the FVIIIpolypeptide used herein comprises a single chain. In one embodiment, thesingle chain Factor VIII contains an intact intracellular processingsite. In another embodiment, the single chain Factor VIII of theinvention comprises a substitution or mutation at an amino acid positioncorresponding to Arginine 1645, a substitution or mutation at an aminoacid position corresponding to Arginine 1648, or a substitution ormutation at amino acid positions corresponding to Arginine 1645 andArginine 1648 in full-length Factor VIII. In other embodiments, theamino acid substituted at the amino acid position corresponding toArginine 1645 is a different amino acid from the amino acid substitutedat the amino acid position corresponding to Arginine 1648. In certainembodiments, the substitution or mutation is an amino acid other thanarginine, e.g., isoleucine, leucine, lysine, methionine, phenylalanine,threonine, tryptophan, valine, alanine, asparagine, aspartic acid,cysteine, glutamic acid, glutamine, glycine, proline, selenocysteine,serine, tyrosine, histidine, ornithine, pyrrolysine, or taurine. Thesingle chain Factor VIII-Fc fusion protein can run at approximately 220kDa on a non reducing SDS-PAGE and at approximately 195 kDa on areducing SDS-PAGE.

The Factor VIII portion in the FVIII polypeptide used herein has FactorVIII activity. Factor VIII activity can be measured by any known methodsin the art. For example, one of those methods can be a chromogenicassay. The chromogenic assay mechanism is based on the principles of theblood coagulation cascade, where activated Factor VIII accelerates theconversion of Factor X into Factor Xa in the presence of activatedFactor IX, phospholipids and calcium ions. The Factor Xa activity isassessed by hydrolysis of a p-nitroanilide (pNA) substrate specific toFactor Xa. The initial rate of release of p-nitroaniline measured at 405nM is directly proportional to the Factor Xa activity and thus to theFactor VIII activity in the sample. The chromogenic assay is recommendedby the Factor VIII and Factor IX Subcommittee of the Scientific andStandardization Committee (SSC) of the International Society onThrombosis and Hemostasis (ISTH). Since 1994, the chromogenic assay hasalso been the reference method of the European Pharmacopoeia for theassignment of FVIII concentrate potency. Thus, in one embodiment, theFVIII polypeptide comprising single chain Factor VIII has Factor VIIIactivity comparable to a FVIII polypeptide comprising processed FactorVIII, when the Factor VIII activity is measured in vitro by achromogenic assay.

In certain embodiment, a long-acting FVII polypeptide is a FVIIImonomer-dimer hybrid. To obtain a FVIII monomer-dimer hybrid, the codingsequence of human recombinant B-domain deleted FVIII was obtained byreverse transcription-polymerase chain reaction (RT-PCR) from humanliver poly A RNA (Clontech) using FVIII-specific primers. The FVIIIsequence includes the native signal sequence for FVIII. The B-domaindeletion was from serine 743 (S743; 2287 bp) to glutamine 1638 (Q1638;4969 bp) for a total deletion of 2682 bp. Then, the coding sequence forhuman recombinant Fc was obtained by RT-PCR from a human leukocyte cDNAlibrary (Clontech) using Fc specific primers. Primers were designed suchthat the B-domain deleted FVII sequence was fused directly to theN-terminus of the Fc sequence with no intervening linker. The FVIIIFcDNA sequence was cloned into the mammalian dual expression vectorpBUDCE4.1 (Invitrogen) under control of the CMV promoter. A secondidentical Fc sequence including the mouse Igk signal sequence wasobtained by RT-PCR and cloned downstream of the second promoter, EF1α,in the expression vector pBUDCE4.1.

In one embodiment, an FcRn binding partner can be an Fc region. An FcRnbinding partner is any molecule that can be specifically bound by theFcRn receptor with consequent active transport by the FcRn receptor ofthe FcRn binding partner. Thus, the term Fc includes any variants of IgGFc that are functional. The region of the Fc portion of IgG that bindsto the FcRn receptor has been described based on X-ray crystallography(Burmeister et al., Nature 372:379 (1994), incorporated herein byreference in its entirety). The major contact area of the Fc with theFcRn is near the junction of the CH2 and CH3 domains. Fc-FcRn contactsare all within a single Ig heavy chain. The FcRn binding partnersinclude, e.g., whole IgG, the Fc fragment of IgG, and other fragments ofIgG that include the complete binding region of FcRn. The major contactsites include amino acid residues 248, 250-257, 272, 285, 288, 290-291,308-311, and 314 of the CH2 domain and amino acid residues 385-387, 428,and 433-436 of the CH3 domain. References made to amino acid numberingof immunoglobulins or immunoglobulin fragments, or regions, are allbased on Kabat et al. 1991, Sequences of Proteins of ImmunologicalInterest, U. S. Department of Public Health, Bethesda; MD, incorporatedherein by reference in its entirety. (The FcRn receptor has beenisolated from several mammalian species including humans. The sequencesof the human FcRn, rat FcRn, and mouse FcRn are known (Story et al., J.Exp. Med 180: 2377 (1994), incorporated herein by reference in itsentirety.) An Fc can comprise the CH2 and CH3 domains of animmunoglobulin with or without the hinge region of the immunoglobulin.Exemplary Fc variants are provided in WO 2004/101740 and WO 2006/074199,incorporated herein by reference in its entirety.

Fc (or Fc portion of a chimeric polypeptide) can contain one or moremutations, and combinations of mutations.

Fc (or Fc portion of a chimeric polypeptide) can contain mutationsconferring increased half-life such as M252Y, S254T, T256E, andcombinations thereof, as disclosed in Oganesyan et al., Mol. Immunol.46:1750 (2009), which is incorporated herein by reference in itsentirety; H433K, N434F, and combinations thereof, as disclosed inVaccaro et al., Nat. Biotechnol. 23:1283 (2005), which is incorporatedherein by reference in its entirety; the mutants disclosed at pages 1-2,paragraph [0012], and Examples 9 and 10 of US 2009/0264627 A1, which isincorporated herein by reference in its entirety; and the mutantsdisclosed at page 2, paragraphs [0014] to [0021] of US 20090163699 A1,which is incorporated herein by reference in its entirety.

Fc (or Fc portion of a chimeric polypeptide) can also include, e.g., thefollowing mutations: The Fc region of IgG can be modified according towell recognized procedures such as site directed mutagenesis and thelike to yield modified IgG or Fc fragments or portions thereof that willbe bound by FcRn. Such modifications include, e.g., modifications remotefrom the FcRn contact sites as well as modifications within the contactsites that preserve or even enhance binding to the FcRn. For example thefollowing single amino acid residues in human IgG1 Fc (Fcy1) can besubstituted without significant loss of Fc binding affinity for FcRn:P238A, S239A, K246A, K248A, D249A, M252A, T256A, E258A, T260A, D265A,S267A, H268A, E269A, D270A, E272A, L274A, N276A, Y278A, D280A, V282A,E283A, H285A, N286A, T289A, K290A, R292A, E293A, E294A, Q295A, Y296F,N297A, S298A, Y300F, R301A, V303A, V305A, T307A, L309A, Q311A, D312A,N315A, K317A, E318A, K320A, K322A, S324A, K326A, A327Q, P329A, A330Q,A330S, P331A, P331S, E333A, K334A, T335A, S337A, K338A, K340A, Q342A,R344A, E345A, Q347A, R355A, E356A, M358A, T359A, K360A, N361A, Q362A,Y373A, S375A D376A, A378Q, E380A, E382A, S383A, N384A, Q386A, E388A,N389A, N390A, Y391F, K392A, L398A, S400A, D401A, D413A, K414A, R416A,Q418A, Q419A, N421A, V422A, S424A, E430A, N434A, T437A, Q438A, K439A,S440A, S444A, and K447A, where for example P238A represents wildtypeproline substituted by alanine at position number 238. In addition toalanine other amino acids can be substituted for the wildtype aminoacids at the positions specified above. Mutations can be introducedsingly into Fc giving rise to more than one hundred FcRn bindingpartners distinct from native Fc. Additionally, combinations of two,three, or more of these individual mutations can be introduced together,giving rise to hundreds more FcRn binding partners. Certain of thesemutations can confer new functionality upon the FcRn binding partner.For example, one embodiment incorporates N297A, removing a highlyconserved N-glycosylation site. The effect of this mutation is to reduceimmunogenicity, thereby enhancing circulating half-life of the FcRnbinding partner, and to render the FcRn binding partner incapable ofbinding to FcyRI, FcyRIIA, FcyRIIB, and FcyRIIIA, without compromisingaffinity for FcRn (Routledge et al. 1995, Transplantation 60:847, whichis incorporated herein by reference in its entirety; Friend et al. 1999,Transplantation 68:1632, which is incorporated herein by reference inits entirety; Shields et al. 1995, J. Biol. Chem. 276:6591, which isincorporated herein by reference in its entirety). Additionally, atleast three human Fc gamma receptors appear to recognize a binding siteon IgG within the lower hinge region, generally amino acids 234-237.Therefore, another example of new functionality and potential decreasedimmunogenicity can arise from mutations of this region, as for exampleby replacing amino acids 233-236 of human IgG1 “ELLG” to thecorresponding sequence from IgG2 “PVA” (with one amino acid deletion).It has been shown that FcyRI, FcyRII, and FcyRIII which mediate variouseffector functions will not bind to IgG when such mutations have beenintroduced (Ward and Ghetie, Therapeutic Immunology 2:77 (1995), whichis incorporated herein by reference in its entirety; and Armour et al.,Eur. J. Immunol. 29:2613 (1999), which is incorporated herein byreference in its entirety). As a further example of new functionalityarising from mutations described above affinity for FcRn can beincreased beyond that of wild type in some instances. This increasedaffinity can reflect an increased “on” rate, a decreased “off” rate orboth an increased “on” rate and a decreased “off” rate. Mutationsbelieved to impart an increased affinity for FcRn include, e.g., T256A,T307A, E380A, and N434A (Shields et al., J. Biol. Chem. 276:6591 (2001),which is incorporated herein by reference in its entirety).

The Fc (or Fc portion of a chimeric polypeptide) can be at least 60%,70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the Fcamino acid sequence shown in Table 11 (amino acids 1458 to 1684 of SEQID NO:2 or amino acids 2352 to 2578 of SEQ ID NO:6). The Fc (or Fcportion of a chimeric polypeptide) can be identical to the Fc amino acidsequence shown in Table 11 (amino acids 1458 to 1684 of SEQ ID NO:2 andamino acids 2352 to 2578 of SEQ ID NO:6).

As a practical matter, whether any particular nucleic acid molecule orpolypeptide is at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical toa nucleotide sequence or polypeptide of the present invention can bedetermined conventionally using known computer programs. In oneembodiment, a method for determining the best overall match between aquery sequence (reference or original sequence) and a subject sequence,also referred to as a global sequence alignment, can be determined usingthe FASTDB computer program based on the algorithm of Brutlag et al.,Comp. App. Biosci. 6:237-245 (1990), which is herein incorporated byreference in its entirety In a sequence alignment the query and subjectsequences are both DNA sequences. An RNA sequence can be compared byconverting U's to T's. The result of said global sequence alignment isin percent identity. In another embodiment, parameters used in a FASTDBalignment of DNA sequences to calculate percent identity are:Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, Joining Penalty=30,Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap SizePenalty 0.05, Window Size=500 or the length of the subject nucleotidesequence, whichever is shorter.

If the subject sequence is shorter than the query sequence because of 5′or 3′ deletions, not because of internal deletions, a manual correctionmust be made to the results. This is because the FASTDB program does notaccount for 5′ and 3′ truncations of the subject sequence whencalculating percent identity. For subject sequences truncated at the 5′or 3′ ends, relative to the query sequence, the percent identity iscorrected by calculating the number of bases of the query sequence thatare 5′ and 3′ of the subject sequence, which are not matched/aligned, asa percent of the total bases of the query sequence. Whether a nucleotideis matched/aligned is determined by results of the FASTDB sequencealignment. This percentage is then subtracted from the percent identity,calculated by the above FASTDB program using the specified parameters,to arrive at a final percent identity score. This corrected score iswhat is used for the purposes of the present invention. Only basesoutside the 5′ and 3′ bases of the subject sequence, as displayed by theFASTDB alignment, which are not matched/aligned with the query sequence,are calculated for the purposes of manually adjusting the percentidentity score.

For example, a 90 base subject sequence is aligned to a 100 base querysequence to determine percent identity. The deletions occur at the 5′end of the subject sequence and therefore, the FASTDB alignment does notshow a matched/alignment of the first 10 bases at 5′ end. The 10unpaired bases represent 10% of the sequence (number of bases at the 5′and 3′ ends not matched/total number of bases in the query sequence) so10% is subtracted from the percent identity score calculated by theFASTDB program. If the remaining 90 bases were perfectly matched thefinal percent identity would be 90%. In another example, a 90 basesubject sequence is compared with a 100 base query sequence. This timethe deletions are internal deletions so that there are no bases on the5′ or 3′ of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by FASTDB is notmanually corrected. Once again, only bases 5′ and 3′ of the subjectsequence which are not matched/aligned with the query sequence aremanually corrected for. No other manual corrections are to made for thepurposes of the present invention.

By a polypeptide having an amino acid sequence at least, for example,95% “identical” to a query amino acid sequence of the present invention,it is intended that the amino acid sequence of the subject polypeptideis identical to the query sequence except that the subject polypeptidesequence can include up to five amino acid alterations per each 100amino acids of the query amino acid sequence. In other words, to obtaina polypeptide having an amino acid sequence at least 95% identical to aquery amino acid sequence, up to 5% of the amino acid residues in thesubject sequence can be inserted, deleted, (indels) or substituted withanother amino acid. These alterations of the reference sequence canoccur at the amino or carboxy terminal positions of the reference aminoacid sequence or anywhere between those terminal positions, interspersedeither individually among residues in the reference sequence or in oneor more contiguous groups within the reference sequence.

As a practical matter, whether any particular polypeptide is at least85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, theamino acid sequences of SEQ ID NO:2 (the factor VIII portion, the Fcportion, individually or together) or 4, or a known factor VIII or Fcpolypeptide sequence, can be determined conventionally using knowncomputer programs. In one embodiment, a method for determining the bestoverall match between a query sequence (reference or original sequence)and a subject sequence, also referred to as a global sequence alignment,can be determined using the FASTDB computer program based on thealgorithm of Brutlag el al., Comp. App. Biosci. 6:237-245(1990),incorporated herein by reference in its entirety. In a sequencealignment the query and subject sequences are either both nucleotidesequences or both amino acid sequences. The result of said globalsequence alignment is in percent identity. In another embodiment,parameters used in a FASTDB amino acid alignment are: Matrix=PAM 0,k-tuple=2, Mismatch Penalty=1, Joining Penalty=20, Randomization GroupLength=0, Cutoff Score=1, Window Size=sequence length, Gap Penalty=5,Gap Size Penalty=0.05, Window Size=500 or the length of the subjectamino acid sequence, whichever is shorter.

If the subject sequence is shorter than the query sequence due to N- orC-terminal deletions, not because of internal deletions, a manualcorrection must be made to the results. This is because the FASTDBprogram does not account for N- and C-terminal truncations of thesubject sequence when calculating global percent identity. For subjectsequences truncated at the N- and C-termini, relative to the querysequence, the percent identity is corrected by calculating the number ofresidues of the query sequence that are N- and C-terminal of the subjectsequence, which are not matched/aligned with a corresponding subjectresidue, as a percent of the total bases of the query sequence. Whethera residue is matched/aligned is determined by results of the FASTDBsequence alignment. This percentage is then subtracted from the percentidentity, calculated by the above FASTDB program using the specifiedparameters, to arrive at a final percent identity score. This finalpercent identity score is what is used for the purposes of the presentinvention. Only residues to the N- and C-termini of the subjectsequence, which are not matched/aligned with the query sequence, areconsidered for the purposes of manually adjusting the percent identityscore. That is, only query residue positions outside the farthest N- andC-terminal residues of the subject sequence.

For example, a 90 amino acid residue subject sequence is aligned with a100 residue query sequence to determine percent identity. The deletionoccurs at the N-terminus of the subject sequence and therefore, theFASTDB alignment does not show a matching/alignment of the first 10residues at the N-terminus. The 10 unpaired residues represent 10% ofthe sequence (number of residues at the N- and C-termini notmatched/total number of residues in the query sequence) so 10% issubtracted from the percent identity score calculated by the FASTDBprogram. If the remaining 90 residues were perfectly matched the finalpercent identity would be 90%. In another example, a 90 residue subjectsequence is compared with a 100 residue query sequence. This time thedeletions are internal deletions so there are no residues at the N- orC-termini of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by FASTDB is notmanually corrected. Once again, only residue positions outside the N-and C-terminal ends of the subject sequence, as displayed in the FASTDBalignment, which are not matched/aligned with the query sequence aremanually corrected for. No other manual corrections are to made for thepurposes of the present invention.

The polynucleotide variants can contain alterations in the codingregions, non-coding regions, or both. In one embodiment, thepolynucleotide variants contain alterations which produce silentsubstitutions, additions, or deletions, but do not alter the propertiesor activities of the encoded polypeptide. In another embodiment,nucleotide variants are produced by silent substitutions due to thedegeneracy of the genetic code. In other embodiments, variants in which5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added in anycombination. Polynucleotide variants can be produced for a variety ofreasons, e.g., to optimize codon expression for a particular host(change codons in the human mRNA to others, e.g., a bacterial host suchas E. coli).

Naturally occurring variants are called “allelic variants,” and refer toone of several alternate forms of a gene occupying a given locus on achromosome of an organism (Genes II, Lewin, B., ed., John Wiley & Sons,New York (1985)). These allelic variants can vary at either thepolynucleotide and/or polypeptide level and are included in the presentinvention. Alternatively, non-naturally occurring variants can beproduced by mutagenesis techniques or by direct synthesis.

Using known methods of protein engineering and recombinant DNAtechnology, variants can be generated to improve or alter thecharacteristics of the polypeptides. For instance, one or more aminoacids can be deleted from the N-terminus or C-terminus of the secretedprotein without substantial loss of biological function. Ron et al., J.Biol. Chem. 268: 2984-2988 (1993), incorporated herein by reference inits entirety, reported variant KGF proteins having heparin bindingactivity even after deleting 3, 8, or 27 amino-terminal amino acidresidues. Similarly, Interferon gamma exhibited up to ten times higheractivity after deleting 8-10 amino acid residues from the carboxyterminus of this protein. (Dobeli el al., J. Biotechnology 7:199-216(1988), incorporated herein by reference in its entirety.)

Moreover, ample evidence demonstrates that variants often retain abiological activity similar to that of the naturally occurring protein.For example, Gayle and coworkers (J. Biol. Chem 268:22105-22111 (1993),incorporated herein by reference in its entirety) conducted extensivemutational analysis of human cytokine IL-la. They used randommutagenesis to generate over 3,500 individual IL-la mutants thataveraged 2.5 amino acid changes per variant over the entire length ofthe molecule. Multiple mutations were examined at every possible aminoacid position. The investigators found that “[m]ost of the moleculecould be altered with little effect on either [binding or biologicalactivity].” (See Abstract.) In fact, only 23 unique amino acidsequences, out of more than 3,500 nucleotide sequences examined,produced a protein that significantly differed in activity fromwild-type.

As stated above, polypeptide variants include, e.g., modifiedpolypeptides. Modifications include, e.g., acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,pegylation (Mei et al., Blood 116:270-79 (2010), which is incorporatedherein by reference in its entirety), proteolytic processing,phosphorylation, prenylation, racemization, selenoylation, sulfation,transfer-RNA mediated addition of amino acids to proteins such asarginylation, and ubiquitination. In some embodiments, Factor VIII ismodified, e.g., pegylated, at any convenient location. In someembodiments, Factor VIII is pegylated at a surface exposed amino acid ofFactor VIII, e.g., a surface exposed cysteine, which can be anengineered cysteine. Id. In some embodiments, modified Factor VIII,e.g., pegylated Factor VIII, is a long-acting Factor VIII.

III. Method of Administering

Treatment of hemophilia A is a replacement therapy targeting restorationof FVIII activity to 1 to 5% of normal levels to prevent spontaneousbleeding (Mannucci, P. M. et al., N. Engl. J. Med. 344:1773-9 (2001),herein incorporated by reference in its entirety).

The present invention also includes methods of administering thepharmaceutical composition of the invention. The present inventionprovides a method of administering a pharmaceutical composition to ahuman subject in need thereof (e.g., human patient), comprisingadministering to the subject a therapeutic dose of a pharmaceuticalcomposition comprising a Factor VIII polypeptide, e.g., a long-actingFVIII polypeptide or a short-acting FVIII polypeptide, e.g., a FactorVIII-Fc polypeptide, or a hybrid of such a polypeptide at a dosinginterval.

In some embodiments, the pharmaceutical composition of the invention isused to reduce or decrease one or more bleeding episodes or frequency(e.g., a bleeding condition) in a subject in need thereof. In otherembodiments, the pharmaceutical composition of the invention is used totreat or prevent a bleeding condition in a subject in need thereof.

The bleeding condition can be caused by a blood coagulation disorder. Ablood coagulation disorder can also be referred to as a coagulopathy. Inone example, the blood coagulation disorder, which can be treated with apharmaceutical composition of the current disclosure, is hemophilia. Inanother example, the blood coagulation disorder, which can be treatedwith a pharmaceutical composition of the present disclosure ishemophilia A.

In some embodiments, the type of bleeding associated with the bleedingcondition is selected from hemarthrosis, muscle bleed, oral bleed,hemorrhage, hemorrhage into muscles, oral hemorrhage, trauma, traumacapitis, gastrointestinal bleeding, intracranial hemorrhage,intra-abdominal hemorrhage, intrathoracic hemorrhage, bone fracture,central nervous system bleeding, bleeding in the retropharyngeal space,bleeding in the retroperitoneal space, and bleeding in the illiopsoassheath.

In other embodiments, the subject suffering from bleeding condition isin need of treatment for surgery, including, e.g., surgical prophylaxisor peri-operative management. In one example, the surgery is selectedfrom minor surgery and major surgery. Exemplary surgical proceduresinclude tooth extraction, tonsillectomy, inguinal herniotomy,synovectomy, craniotomy, osteosynthesis, trauma surgery, intracranialsurgery, intra-abdominal surgery, intrathoracic surgery, jointreplacement surgery (e.g., total knee replacement, hip replacement, andthe like), heart surgery, and caesarean section.

The present invention also provides a method of reducing or decreasingan annualized bleeding rate (ABR) of a subject having hemophiliacomprising administering to the subject an effective dose of a FVIIIpolypeptide. In one embodiment, a long-acting FVIII polypeptide isadministered at a dosing interval of every three days or longer. Inanother embodiment, the effective dose is between about 20 IU/kg andabout 90 IU/kg. In other embodiments, the effective dose is 20-30 IU/kg,30-40 IU/kg, 40-50 IU/kg, 50-60 IU/kg, 60-70 IU/kg, 70-80 IU/kg, or80-90 IU/kg. In yet other embodiments, the effective dose is 20 IU/kg,25 IU/kg, 30 IU/kg, 35 IU/kg, 40 IU/kg, 45 IU/kg, 50 IU/kg, 55 IU/kg, 60IU/kg, 65 IU/kg, 70 IU/kg, 75 IU/kg, 80 IU/kg, 85 IU/kg, or 90 IU/kg.

In certain embodiments, administration of a FVIII polypeptide is forindividualized (tailored) prophylaxis and results in an ABR of less thanabout 5.5, less than about 5.4, less than about 5.3, less than about5.2, less than about 5.1, less than about 5.0, less than about 4.9, lessthan about 4.8, less than about 4.7, less than about 4.6, or less thanabout 4.5. In other embodiments, the administration results in an ABR ofabout 4.7 to 0. In some embodiments, the median of the ABR is about 1.6.In yet other embodiments, the mean of the ABR is about 2.9. In oneaspect of the individualized prophylaxis regimen, the effective dose isabout 25 IU/kg to about 65 IU/kg given every three to five days. Forexample, the effective dose is about 25 IU/kg to about 65 IU/kg givenevery three days. In another aspect, the effective dose is about 50IU/kg to about 65 IU/kg every four days or every five days. In otheraspects, the effective dose is up to 65 IU/kg every three days. In stillother embodiments, the effective dose of a FVIII polypeptide is modifiedbased on the patient's pharmacokinetic profile. In one example, thepatent is administered with two doses initially, a first dose of 25IU/kg on day 1 and a second dose of 50 IU/kg on day 4. If thepharmacokinetic data show less than 1% trough level of normal FVIIIactivity after the initial two doses, the patient is then administeredwith about 25 IU/kg to about 65 IU/kg every three days. In anotherexample, if the pharmacokinetic data show higher than 1% trough level ofnormal FVIII activity for five days from the dosing, the patient is thenadministered with about 50 IU/kg to about 65 IU/kg every five days. Ifthe patient experiences more than two spontaneous bleeds over eightweeks period after administration of about 50 IU/kg to about 65 IU/kgevery five days, the patient is administered one of the followings: (1)up to about 65 IU/kg every three days (for the target trough level of upto 5% of normal), (2) about 50 IU/kg to about 65 IU/kg every four days(for the target trough level up to 5% of normal), or (3) about 25 IU/kgto about 65 IU/kg every three days (for the target trough level of 1% to3% of normal). If the patient receiving about 50 IU/kg to about 65 IU/kgevery four days still experiences more than two spontaneous bleeds, themaximum dose can go up to about 65 IU/kg every three days. In someexamples, the Interquartile Range (IQR) of the ABR for theindividualized prophylaxis regimen is 0 to 4.7.

In some embodiments, administration of a FVIII polypeptide is for weeklyprophylaxis and results in an ABR of less than about 9.0, less thanabout 8.9, less than about 8.8, less than about 8.7, less than about8.6, less than about 8.5, or less than about 8.4. In one example, theadministration results in an ABR between about 8.4 and 0. In anotherexample, the median of the ABR for weekly prophylaxis is about 3.6. Inother examples, the mean of the ABR for weekly prophylaxis is about 8.8.In other embodiments, an effective dose for weekly prophylaxis is about65 IU/kg once every week, e.g., every five days, every six days, everyseven days, every eight days, or every nine days. In some examples, theIQR of the ABR for the weekly prophylaxis regimen is 1.9 to 8.4.

In other embodiments, administration of a FVIII polypeptide is forepisodic or on-demand treatment and results in an ABR of less than about55, less than about 54, less than about 53, less than about 52, lessthan about 51, less than about 50, less than about 49, less than about48, or less than about 47. In one example, the administration results inan ABR between about 49 and 0, e.g., between 48.7 and 0. In anotherexample, the median of the ABR for episodic (on-demand) treatment isabout 33.5. In other examples, the mean of the ABR for episodic(on-demand) treatment is about 37.23. In other embodiments, an effectivedose for on-demand treatment is about 10 IU/kg to 75 IU/kg every 12 to24 hours. In some examples, the IQR of the ABR for the on-demandtreatment is 21.1 to 48.7.

In some embodiments, the effective dose for individualized prophylaxis,weekly prophylaxis, or episodic treatment is a fixed dose or astratified dose. In one aspect, the fixed dose is about 2,000 IU perdose, about 2,500 IU per dose, about 3,000 IU per dose, about 3,500 IUper dose, or about 4,000 IU per dose.

The dosing interval for a long-acting FVIII polypeptide can be at leastabout one and one-half to six times longer, one and one-half to fivetimes longer, one and one-half to four times longer, one and one-half tothree times longer, or one and one-half to two times longer, than thedosing interval required for an equivalent dose of the Factor VIIIwithout the non-Factor VIII portion (a polypeptide consisting of theFactor VIII portion), e.g., without the Fc portion. The dosing intervalcan be at least about one and one-half, two, two and one-half, three,three and one-half, four, four and one-half, five, five and one-half orsix times longer than the dosing interval required for an equivalentdose of the Factor VIII without the non-Factor VIII portion (apolypeptide consisting of the Factor VIII portion), e.g., without the Fcportion. The dosing interval can be about every three, four, five, six,seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen days orlonger.

The methods of the invention can be practiced on a subject in need ofprophylactic treatment or on-demand treatment.

For on-demand treatment, the dosing interval of a pharmaceuticalcomposition comprising a FVIII polypeptide is about once every 24-36,24-48, 24-72, 24-96, 24-120, 24-144, 24-168, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, or 72 hours or longer.

In one embodiment, the subject is in need of on-demand (episodic)treatment. In another embodiment, on-demand (episodic) treatmentresolves greater than 80% (greater than 80%, greater than 81%, greaterthan 82%, greater than 83%, greater than 84%, greater than 85%, greaterthan 86%, greater than 87%, greater than 88%, greater than 89%, greaterthan 90%, greater than 91%, greater than 92%, greater than 93%, greaterthan 94%, greater than 95%, greater than 96%, greater than 97%, greaterthan 98%, greater than 99%, or 100%) or 80-100%, 80-90%, 85-90%,90-100%, 90-95%, or 95-100% of bleeds (e.g., spontaneous bleeds) in asingle dose. In another embodiment, greater than 80% (greater than 81%,greater than 82%, greater than 83%, greater than 84%, greater than 85%,greater than 86%, greater than 87%, greater than 88%, greater than 89%,greater than 90%, greater than 91%, greater than 92%, greater than 93%,greater than 94%, greater than 95%, greater than 96%, greater than 97%,greater than 98%, or 100%) or 80-100%, 80-90%, 85-90%, 90-100%, 90-95%,or 95-100% of bleeding episodes are rated excellent or good byphysicians after on-demand (episodic) treatment. In other embodiments,greater than 5%, (greater than 6%, greater than 7%, greater than 8%,greater than 9%, greater than 10%, greater than 11%, greater than 12%,greater than 13%, greater than 14%, greater than 15%, greater than 16%,greater than 17%, greater than 18%, greater than 19%, greater than 20%),or 5-20%, 5-15%, 5-10%, 10-20%, or 10-15% of bleeding episodes are ratedas fair by physicians after on-demand treatment.

In other embodiments, the pharmaceutical composition comprising a FVIIIpolypeptide is used for prophylaxis. Prophylaxis can be demonstrated bybetter Cmax, better Tmax, and/or greater mean residence time versusshort-acting FVIII. In some embodiments, prophylaxis results in nospontaneous bleeding episodes within about 24, 36, 48, 72, or 96 hours(e.g., 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 96, 87, 88, 89, 90, 91, 92, 93, 94,95, or 96 hours), after injection (e.g., the last injection). In certainembodiments, prophylaxis results in greater than 30% (e.g., greater than31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 96, 87, 88, 89, or 90%, for example, greater than 50%), meanreduction in annualized bleeding episodes with once weekly dosing (e.g.,at 65 IU/kg).

In another example, the subject is concomitantly treated with FIX.Because the compounds of the invention are capable of activating FIXa,they could be used to pre-activate the FIXa polypeptide beforeadministration of the FIXa to the subject.

IV. Method of Making

A FVIII polypeptide can be manufactured in a host cell comprising avector encoding the FVIII polypeptide. In one embodiment, the host cellis transformed with one or more vectors comprising a first nucleotidesequence encoding a FVIII polypeptide and a first FcRn polypeptide, asecond nucleotide sequence encoding a second FcRn polypeptide, andoptionally a third nucleotide sequence encoding a protein convertase,e.g., PC5. As used herein, an expression vector refers to any nucleicacid construct which contains the necessary elements for thetranscription and translation of an inserted coding sequence, or in thecase of an RNA viral vector, the necessary elements for replication andtranslation, when introduced into an appropriate host cell. Expressionvectors can include plasmids, phagemids, viruses, and derivativesthereof.

A gene expression control sequence as used herein is any regulatorynucleotide sequence, such as a promoter sequence or promoter-enhancercombination, which facilitates the efficient transcription andtranslation of the coding nucleic acid to which it is operably linked.The gene expression control sequence can, for example, be a mammalian orviral promoter, such as a constitutive or inducible promoter.Constitutive mammalian promoters include, but are not limited to, thepromoters for the following genes: hypoxanthine phosphoribosyltransferase (HPRT), adenosine deaminase, pyruvate kinase, beta-actinpromoter, and other constitutive promoters. Exemplary viral promoterswhich function constitutively in eukaryotic cells include, for example,promoters from the cytomegalovirus (CMV), simian virus (e.g., SV40),papilloma virus, adenovirus, human immunodeficiency virus (HIV), Roussarcoma virus, cytomegalovirus, the long terminal repeats (LTR) ofMoloney leukemia virus, and other retroviruses, and the thymidine kinasepromoter of herpes simplex virus. Other constitutive promoters are knownto those of ordinary skill in the art. The promoters useful as geneexpression sequences of the invention also include inducible promoters.Inducible promoters are expressed in the presence of an inducing agent.For example, the metallothionein promoter is induced to promotetranscription and translation in the presence of certain metal ions.Other inducible promoters are known to those of ordinary skill in theart.

Examples of vectors include, but are not limited to viral vectors orplasmid vectors. Plasmid vectors have been extensively described in theart and are well-known to those of skill in the art. See, e.g., Sambrooket al., Molecular Cloning: A Laboratory Manual, Second Edition, ColdSpring Harbor Laboratory Press, 1989. In the last few years, plasmidvectors have been found to be particularly advantageous for deliveringgenes to cells in vivo because of their inability to replicate withinand integrate into a host genome. These plasmids, however, having apromoter compatible with the host cell, can express a peptide from agene operably encoded within the plasmid. Some commonly used plasmidsavailable from commercial suppliers include pBR322, pUC18, pUC19,various pcDNA plasmids, pRC/CMV, various pCMV plasmids, pSV40, andpBlueScript. Additional examples of specific plasmids include pcDNA3.1,catalog number V79020; pcDNA3.1/hygro, catalog number V87020;pcDNA4/myc-His, catalog number V86320; and pBudCE4.1, catalog numberV53220, all from Invitrogen (Carlsbad, Calif.). Other plasmids arewell-known to those of ordinary skill in the art. Additionally, plasmidscan be custom designed using standard molecular biology techniques toremove and/or add specific fragments of DNA.

The expression vector or vectors are then transfected or co-transfectedinto a suitable target cell, which will express the polypeptides.Transfection techniques known in the art include, but are not limitedto, calcium phosphate precipitation (Wigler et al. (1978) Cell 14:725),electroporation (Neumann et al. (1982) EMBO J 1:841), and liposome-basedreagents. A variety of host-expression vector systems can be utilized toexpress the proteins described herein including both prokaryotic andeukaryotic cells. These include, but are not limited to, microorganismssuch as bacteria (e.g., E. coli) transformed with recombinantbacteriophage DNA or plasmid DNA expression vectors containing anappropriate coding sequence; yeast or filamentous fungi transformed withrecombinant yeast or fungi expression vectors containing an appropriatecoding sequence; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing an appropriate codingsequence; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus or tobacco mosaic virus) ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid) containing an appropriate coding sequence; or animal cellsystems, including mammalian cells (e.g., HEK 293, CHO, Cos, HeLa,HKB11, and BHK cells).

In one embodiment, the host cell is a eukaryotic cell. As used herein, aeukaryotic cell refers to any animal or plant cell having a definitivenucleus. Eukaryotic cells of animals include cells of vertebrates, e.g.,mammals, and cells of invertebrates, e.g., insects. Eukaryotic cells ofplants specifically can include, without limitation, yeast cells. Aeukaryotic cell is distinct from a prokaryotic cell, e.g., bacteria.

In certain embodiments, the eukaryotic cell is a mammalian cell. Amammalian cell is any cell derived from a mammal. Mammalian cellsspecifically include, but are not limited to, mammalian cell lines. Inone embodiment, the mammalian cell is a human cell. In anotherembodiment, the mammalian cell is a HEK 293 cell, which is a humanembryonic kidney cell line. HEK 293 cells are available as CRL-1533 fromAmerican Type Culture Collection, Manassas, Va., and as 293-H cells,Catalog No. 11631-017 or 293-F cells, Catalog No. 11625-019 fromInvitrogen (Carlsbad, Calif.). In some embodiments, the mammalian cellis a PER.C6® cell, which is a human cell line derived from retina.PER.C6® cells are available from Crucell (Leiden, The Netherlands). Inother embodiments, the mammalian cell is a Chinese hamster ovary (CHO)cell. CHO cells are available from American Type Culture Collection,Manassas, Va. (e.g., CHO-Ki; CCL-61). In still other embodiments, themammalian cell is a baby hamster kidney (BHK) cell. BHK cells areavailable from American Type Culture Collection, Manassas, Va. (e.g.,CRL-1632). In some embodiments, the mammalian cell is a HKB11 cell,which is a hybrid cell line of a HEK293 cell and a human B cell line.Mei et al., Mol. Biotechnol. 34(2): 165-78 (2006).

The method can further comprise purification steps. Various knownpurifications steps are well known in the art.

EXAMPLES Example 1. Product Description

rFVIIIFc is a long-acting, fully recombinant fusion protein consistingof human coagulation Factor VIII (FVIII) covalently linked to the Fcdomain of human immunoglobulin G1 (IgG1). The Factor VIII portion ofrFVIIIFc has a primary amino acid sequence and post-translationalmodifications that are comparable to the 90+80 kDa form of Factor VIII(i.e., B-domain deleted). The Fc domain of rFVIIIFc contains the hinge,CH2 and CH3 regions of IgG1. rFVIIIFc contains 1882 amino acids with anapparent molecular weight of approximately 220 kilodaltons.

rFVIIIFc is produced by recombinant DNA technology in a human embryonickidney (HEK) cell line, which has been extensively characterized. Thecell line expresses rFVIIIFc into a defined cell culture medium thatdoes not contain any proteins derived from animal or human sources.rFVIIIFc is purified by a series of chromatography steps that does notrequire use of a monoclonal antibody. The process includes a detergentviral inactivation step and multiple viral clearance steps including anaffinity chromatography step and a 15 nm virus-retaining nano-filtrationstep. No human or animal additives are used in the cell culture,purification, and formulation processes.

rFVIIIFc is in the pharmacotherapeutic group: antihemorrhagics, bloodcoagulation factor VIII. It is provided as a sterile, preservative-free,non-pyrogenic, lyophilized, white to off-white powder to cake, forintravenous (IV) administration in a single-use vial, accompanied by aliquid diluent in a pre-filled syringe. In addition to rFVIIIFc, thepharmaceutical composition comprises in the lyophilizate sucrose, sodiumchloride, L-Histidine, calcium chloride, and Polysorbate 20 orpolysorbate 80, and comprising in sterilized water for injections. Eachsingle-use vial contains nominally 250, 500, 750, 1000, 1500, 2000,3000, 4000, 5000, or 6000 International Units (IU) of rFVIIIFc. Whenreconstituted with provided diluent, the product contains the followingexcipients: sucrose, sodium chloride, L-histidine, calcium chloride, andpolysorbate 20 or polysorbate 80, at the concentrations shown in Table 1or Table 2 below. The pharmaceutical composition is formulated forintravenous administration only after reconstitution.

Each pack contains a powder vial (type 1 glass) with a stopper (butyl)and a flip-off seal (aluminum), 3 ml solvent in a pre-filled syringe(type I glass) with a plunger stopper (butyl), a tip-cap (butyl), and asterile vial adapter reconstitution device.

TABLE 1 rFVIIIFc Formulations Following Reconstitution with LiquidDiluent Calcium rFVIIIFc Sucrose NaCl L-histidine chloride Polysorbate-20 IU/ml* % (w/v) (mM) (mM) (mM) % (w/v) 83 IU/ml 1.33 205 6.64 5.40.013 167 IU/ml 1.33 205 6.64 5.4 0.013 250 IU/ml 1.33 205 6.64 5.40.013 333 IU/ml 1.33 205 6.64 5.4 0.013 500 IU/ml 1.33 205 6.64 5.40.013 667 IU/ml 1.33 205 6.64 5.4 0.013 1000 IU/ml 1.33 205 6.64 5.40.013 1333 IU/ml 1.33 205 6.64 5.4 0.013 1667 IU/ml 1.33 205 6.64 5.40.013 2000 IU/ml 1.33 205 6.64 5.4 0.013

TABLE 2 rFVIIIFc Formulations Following Reconstitution with LiquidDiluent Concentration 250 500 750 1000 1500 2000 3000 4000 5000 6000Component IU/vial IU/vial IU/vial IU/vial IU/vial IU/vial IU/vialIU/vial IU/vial IU/vial rFVIIIFc* 83 167 250 333 500 667 1000 1333 16672000 IU/mL IU/mL IU/mL IU/mL IU/mL IU/mL IU/mL IU/mL IU/mL IU/mL Sucrose13.3 13.3 13.3 13.3 13.3 13.3 13.3 13.3 13.3 13.3 mg/mL mg/mL mg/mLmg/mL mg/mL mg/mL mg/mL mg/mL mg/mL mg/mL Sodium Chloride 12.0 12.0 12.012.0 12.0 12.0 12.0 12.0 12.0 12.0 mg/mL mg/mL mg/mL mg/mL mg/mL mg/mLmg/mL mg/mL mg/mL mg/mL L-Histidine 1.03 1.03 1.03 1.03 1.03 1.03 1.031.03 1.03 1.03 mg/mL mg/mL mg/mL mg/mL mg/mL mg/mL mg/mL mg/mL mg/mLmg/mL Calcium Chloride 0.80 0.80 0.80 0.80 0.80 0.80 0.80 0.80 0.80 0.80Dihydrate mg/mL mg/mL mg/mL mg/mL mg/mL mg/mL mg/mL mg/mL mg/mL mg/mLPolysorbate 20 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13 mg/mLmg/mL mg/mL mg/mL mg/mL mg/mL mg/mL mg/mL mg/mL mg/mL

Example 2. Method of Formulation

The rFVIIIFc drug product is a sterile lyophilized powder for injectionintended for intravenous administration. Itis supplied in a asepticallyfilled single use vials which contain nominally 50, 500, 750, 1000,1500, 2000, 3000, 4000, 5000 and 6000 IU per vial. The vials are 10 mLUSP/Ph. Eur. Type 1 glass vials sealed with a 20 mm Teflon-coated butylrubber lyophilization stopper and aluminum flip-off crimp seal. Prior tolyophilization, the nominal fill volume target for 250 through 6000 IUvials is 3 mL. The composition of the formulation excipients prior tolyophilization is the same for all dosage strengths. The powder forinjection is reconstituted with 3 mL of diluent comprising sterilizedwater for injections supplied in a sterile prefilled syringe.

The compositions of the drug product solutions prior to lyophilizationare presented in Table 3. The compositions of the drug productsfollowing reconstitution are presented in Table 1 or in Table 2.(Example 1).

TABLE 3 Pre-Iyophilization Concentrations of rFVIIIFc FormulationComponents Concentration 250 500 750 1000 1500 2000 3000 4000 5000 6000Component IU/vial IU/vial IU/vial IU/vial IU/vial IU/vial IU/vialIU/vial IU/vial IU/vial rFVIIIFe 150 287.5 431.25 575 862.5 1150 17252300 2875 3450 IU/ml* IU/ml** IU/ml** IU/ml** IU/ml** IU/ml** IU/ml**IU/ml** IU/ml** IU/ml** Sucrose 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.020.0 20.0 mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml mg/mlSodium 18.0 18.0 18.0 18.0 18.0 18.0 18.0 18.0 18.0 18.0 Chloride mg/mlmg/ml mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml L-Histidine 1.551.55 1.55 1.55 1.55 1.55 1.55 1.55 1.55 1.55 mg/ml mg/ml mg/ml mg/mlmg/ml mg/ml mg/ml mg/ml mg/ml mg/ml Calcium 1.18 1.18 1.18 1.18 1.181.18 1.18 1.18 1.18 1.18 Chloride mg/ml mg/ml mg/ml mg/ml mg/ml mg/mlmg/ml mg/ml mg/ml mg/ml Dihydrate Polysorbate 0.2 0.2 0.2 0.2 0.2 0.20.2 0.2 0.2 0.2 20 mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml mg/mlmg/ml Water for To make 1 ml *** Injection Fill Volume 2.0 ml 2.0 ml 2.0ml 2.0 ml 2.0 ml 2.0 ml 2.0 ml 2.0 ml 2.0 ml 2.0 ml *A 20% overage isincluded for the 250 IU/vial strength to ensure that, after process andtesting variations, at least the nominal concentration is present in thevial. **A 15% overage is included for the 500-6000 IU/vial strengths toensure that, after process and testing variations, at least the nominalconcentration is present in the vial. *** Removed during lyophilization.

Example 3. Dosage and Method of Administration/Method of CalculatingInitial Estimated Dose

rFVIIIFc is long-acting anti-hemophilic factor (recombinant) indicatedin adults and children (≥12 years) with hemophilia A (congenital FactorFVIII deficiency) for, e.g., control and prevention of bleedingepisodes, routine prophylaxis to prevent or reduce the frequency ofbleeding episodes, and perioperative management (surgical prophylaxis).

Dosing of rFVIIIFc can be estimated as described in this example, butcan also be determined by standard tests such as FVIII activity assaysdescribed elsewhere herein.

1 IU of rFVIIIFc per kg body weight is expected to increase thecirculating level of Factor VIII by 2 [IU/dL]. rFVIIIFc has been shownto have a prolonged circulating half-life.

Since patients can vary in their pharmacokinetic (e.g., half-life, invivo recovery) and clinical responses to rFVIIIFc, the expected in vivopeak increase in Factor VIII level expressed as IU/dL (or % of normal)or the required dose can be estimated using the following formulas:

IU/dL (or % of normal)=[Total Dose (IU)/body weight (kg)]×2 (IU/dL perIU/kg)

OR

Dose (IU)=body weight (kg)×Desired Factor VIII Rise (IU/dL or % ofnormal)×0.5 (IU/kg per IU/dL)

The following table (Table 4) can be used to guide dosing in bleedingepisodes:

TABLE 4 Guide to rFVIIIFc Dosing for Treatment of Bleeding DesiredFactor VIII Dose Level (IU/kg)/ (IU/dL or Frequency of Severity of Bleed% of normal) Doses (hrs) Minor and Moderate 40-60  20-30 IU/kg Forexample: joint, Repeat every 24-48 hours superficial muscle/no untilbleeding is resolved neurovascular compromise (except iliopsoas), deeplaceration and renal, superficial soft tissue, mucous membranes Major80-100 40-50 IU/kg For example: iliopsoas Repeat every 12-24 hours anddeep muscle with until bleeding is resolved neurovascular injury, orsubstantial blood loss, retroperitoneum, CNS, throat and neck,gastrointestinal. Adapted from WFH 2012

Subsequent dosage and duration of treatment depends on the individualclinical response, the severity of the Factor VIII deficiency, and thelocation and extent of bleeding.

The following table (Table 5) can be used to guide dosing forperioperative management (surgical prophylaxis):

TABLE 5 Guide to rFVIIIFc Dosing for Perioperative Management (SurgicalProphylaxis) Target Factor VIII Level Dose (IU/kg)/ (IU/dL or FrequencyType of Surgery % of normal) of Doses (hrs) Minor 50 to 80  25-40 IU/kgMinor operations A single infusion can be including uncomplicatedsufficient. Repeat every dental extraction 24 hours as needed to controlbleeding. Major 80 to 120 An initial preoperative Major operations doseof 40-60 IU/kg including intra- followed by a repeat dose abdominaljoint of 40-50 IU/kg after 8-24 replacement surgery hours and then every24 hours to maintain FVIII activity within the target range. rFVIIIFchas a longer half-life than plasma and recombinant FVIII products (seeExample 5)

For routine prophylaxis, the recommended regimen is 50 IU/kg every 3-5days. The dose can be adjusted based on patient response in the range of25-65 I/kg.

For weekly prophylaxis, the recommended dose is 65 IU/kg.

rFVIIIFc is contraindicated in patients who have manifested severehypersensitivity reactions, including anaphylaxis, to the product or itscomponents. Severe hypersensitivity reactions were not observed inclinical trials; however, these have been known to occur with use ofother factor VIII replacement factors.

The clinical response to rFVIIIFc can vary. If bleeding is notcontrolled with the recommended dose, the plasma level of Factor VIIIcan be determined, and a sufficient dose of rFVIIIFc can be administeredto achieve a satisfactory clinical response. If the patient's plasmaFactor VIII level fails to increase as expected or if bleeding is notcontrolled after rFVIIIFc administration, the presence of an inhibitor(neutralizing antibodies) should be suspected, and appropriate testingperformed. Patients using rFVIIIFc can be monitored for the developmentof Factor VIII inhibitors by appropriate clinical observations andlaboratory tests known to those of ordinary skill in the art.

Patient's plasma can be monitored for Factor VIII activity levels, e.g.,the one-stage clotting assay to confirm adequate Factor VIII levels havebeen achieved and maintained, when clinically indicated. Patient'splasma can further be monitored for the development of Factor VIIIinhibitors.

Example 4. Phase 3 Clinical Trial of Extended Half-Life Recombinant FcFusion Factor VIII (“A-LONG” Study)

Summary of A-LONG Study

In the A-LONG study, 165 male patients aged 12 years of age and olderwere enrolled. The A-LONG study had three treatment arms: individualizedprophylaxis, weekly prophylaxis and episodic (on-demand) treatment (Arms1, 2 and 3, respectively). In a subgroup of patients across treatmentarms, rFVIIIFc was evaluated in the perioperative management of patientswho required a major surgical procedure during the study.

Overall, 92.7 percent of patients completed the study. RecombinantFVIIIFc was generally well-tolerated. No inhibitors to rFVIIIFc weredetected and no cases of anaphylaxis were reported in any patients, allof whom switched from commercially-available Factor VIII products. Noserious adverse events were assessed to be related to drug by theinvestigator.

The most common adverse events (incidence of ≥5 percent) occurringoutside of the perioperative management period were nasopharyngitis,arthralgia, headache, and upper respiratory tract infection.

The median (mean) annualized bleeding rates (ABR), including spontaneousand traumatic bleeds, were 1.6 (2.9) in the individualized prophylaxisarm, 3.6 (8.8) in the weekly prophylaxis arm and 33.6 (37.2) in theepisodic treatment arm. In the individualized prophylaxis arm, themedian dosing interval was 3.5 days. During the last 3 months on study,30 percent of patients in the individualized prophylaxis arm achieved amean dosing interval of at least 5 days.

Control of bleeding was assessed in all patients who experienced ableeding episode during the study. Overall, 98 percent of bleedingepisodes were controlled by one or two injections of rFVIIIFc.

In addition, rFVIIIFc was assessed in the perioperative management of 9patients undergoing 9 major surgical procedures. The treating physiciansrated the hemostatic efficacy of rFVIIFc as excellent or good in 100percent of these surgeries.

A-LONG included pharmacokinetic (PK) analysis of rFVIIIFc in allpatients in the study. In a protocol-defined subset of patients withextensive PK sampling, the approximate terminal half-life of rFVIIIFcwas 19.0 hours compared to 12.4 hours for ADVATE® [Antihemophilic Factor(Recombinant), Plasma/Albumin-Free Method], consistent with the resultsobtained in the Phase 1/2 study of rFVIIIFc.

About the A-LONG Study and the rFVIIIFc Program

A-LONG was a global, open-label, multi-center Phase 3 study thatevaluated the efficacy, safety and pharmacokinetics ofintravenously-injected rFVIIIFc. The study was designed to evaluaterFVIIIFc in the control and prevention of bleeding, routine prophylaxisand perioperative management in patients with hemophilia A. A-LONGinvolved 60 hemophilia treatment centers in 19 countries on 6continents.

The A-LONG study had three treatment arms. In Arm 1 (individualizedprophylaxis; n=117), patients were treated with 25-65 IU/kg of rFVIIIFc,at an interval of every three to five days, which was individualized tomaintain factor trough levels sufficient to prevent bleeding. In Arm 2(weekly prophylaxis; n=24), patients were treated with a weekly dose of65 IU/kg. In Arm 3 (episodic treatment; n=23), patients receivedrFVIIIFc episodic treatment as needed for bleeding. In a subgroup ofpatients across treatment arms, rFVIIIFc was evaluated in the surgicalsetting.

The primary efficacy measures were the per-patient annualized bleedingrate in Arm 1 vs. Arm 3, and pharmacokinetics of rFVIIIFc vs. rFVIII(one-stage clotting [activated partial thromboplastin time] assay andthe chromogenic assays calibrated to normal human references plasma withpotency traceable to Word Health Organization standards). The safetyendpoints included the incidence of adverse events and inhibitordevelopment (Nijmegen-modified Bethesda assay) in patients studied forup to 54 weeks. Secondary efficacy endpoints included ABR in Arm 2 vs.Arm 3, annualized number of spontaneous and joint bleeding episodes perpatient, number of injections and dose per injections to resolve ableed, and investigators' assessments of patients' response to surgerywith rFVIIIFc using a bleeding response scale (surgery subgroup only),response to treatment of bleeding episodes and the pharmacokinetics ofrFVIIIFc versus ADVATE®. Non-neutralizing antibodies (NNAs) wereassessed with an electrochemiluminescence-based anti-rFVIIIFcbinding-antibody assay.

Ongoing clinical studies of rFVIIIFc include the Kids A-LONG and ASPIREstudies. Kids A-LONG is a Phase 3, open-label study inpreviously-treated children with hemophilia A under age 12, which isactively recruiting patients. ASPIRE is a long-term open-label study forpatients who completed the A-LONG study or who complete the Kids A-LONGstudy.

A-LONG Study Design

Design: Global, open-label, multicenter, Phase 3 study. The studyprotocol was approved by local Institutional Review Boards for eachparticipating institution, and the study was conducted in accordancewith the International Conference on Harmonisation guidelines for GoodClinical Practice.

Objectives: To evaluate the efficacy and safety ofintravenously-injected recombinant factor VIII Fc fusion protein(rFVIIlFc) in the control and prevention of bleeding episodes, routineprophylaxis, and perioperative management in individuals with severehemophilia A.

Key Inclusion Criteria:

-   -   a. Male    -   b. ≥212 years of age and at least 40 kg    -   c. Diagnosis of severe hemophilia A defined as <1% (<1 IU/dL)        endogenous factor VIII (FVIII) activity    -   d. Previously Treated Patients (PTPs); history of ≥150 prior        documented exposure days (EDs) with any currently marketed FVIII        product, been on prophylaxis ≥2 times per week with a FVIII        product (for Arm 1 only), or experienced ≥12 bleeds in the past        12 months on an episodic regimen.    -   e. No current/prior FVIII inhibitors of measurable activity.    -   f. No history of inhibitors, or history of hypersensitivity or        anaphylaxis associated with any FVIII or intravenous        immunoglobulin administration    -   g. All subjects (or their guardians in the case of minors) gave        informed written consent prior to study participation.

Treatment arms: Details of A-LONG study design is described below.

Arm 1 (individualized prophylaxis) was administered 25-65 IU/kg every3-5 days (maximum 5-day dosing interval). Subjects were treated with aninitial dose of 25 IU/kg on Day 1 and 50 IU/kg on Day 4, which wassubsequently adjusted to maintain trough factor levels sufficient toprevent bleeding. Further dose adjustments to target trough levels of 3to 5 IU/dL permitted after week 7 if subject experienced 22moderate/severe bleeds over a rolling 8-week period; 10 to 20 IU/kgrFVIIIFc (target 20-40 IU/dL FVIII) for minor bleeding episodes; 15 to30 IU/kg rFVIIIFc (target 30-60 IU/dL FVIII) for moderate to majorbleeding episodes; 40 to 50 IU/kg rFVIIIFc (target 80-100 IU/dL FVIII)for major to life-threatening bleeding episodes.

Arm 2 (weekly prophylaxis) was administered 65 IU/kg dose. Subjects weretreated with 65 IU/kg once weekly with no dose or interval adjustment.

Arm 3 (episodic [on-demand] treatment) was administered 10-50 IU/kg.Subjects received rFVIIIFc episodic treatment as needed for bleeding.

A Perioperative Management Subgroup was established. In this subgroup,rFVIIIFc was administered prior to and following surgery in the subsetof patients requiring a major surgical procedure during the study.Subjects in any treatment arm could be enrolled in the surgery subgroup.Eligibility: required major surgery; 12 exposure days to rFVIIIFc withnegative inhibitor titre following this period and within 4 weeks priorto surgery; and, completed, at minimum, abbreviated PK sampling.

All subjects (excluding sequential PK subgroup) underwent PK samplingfrom pre-injection with rFVIIIFc up to 96-hours post-injection,according to schedule: pre-injection, 30 (+3) minutes, 3 hours (15minutes), 72 (+2) hours (Day 3), and 96 (+2) hours (Day 4) from start ofinjection.

Pharmacokinetic (PK) Assessment was performed. All subjects in all armshad an initial PK assessment after their first dose of rFVIIIFc. Asubset of subjects from Arm 1 were assigned to a protocol-specifiedsequential PK subgroup to compare the stability of PK properties ofrFVIIIFc over time with that of recombinant factor VIII (rFVIII, ADVATE®[anti-hemophilic factor (recombinant) plasma/albumin-free method],octocog alfa) as follows:

Prior to treatment in Arm 1, PK was assessed after a single dose ofADVATE® 50 IU/kg. PK was then assessed in these same subjects after asingle dose of rFVIIIFc 50 IU/kg.

PK of rFVIIIFc was repeated at 12 to 24 weeks.

Details on the design of the sequential PK subgroup (Arm 1) dosing andPK sampling is described in FIG. 1

Key Efficacy Outcome Measures (Included in Initial Readout):

-   -   a. Annualized bleeding rate (ABR) in Arm 1 versus Arm 3    -   b. Individualized prophylaxis arm compared with episodic        treatment arm.    -   c. Number of injections required to resolve a bleeding episode    -   d. Number of injections required to resolve bleeding episodes    -   e. Median dose required to resolve bleeding episodes    -   f. Treating physicians' assessments of subjects' response to        surgery with rFVIIIFc using a 4-point scale

PK outcome measures included:

-   -   a. The primary PK assessment were based on FVIII activity levels        determined at a central laboratory by one-stage clotting assay        and by the chromogenic assay against commercially available        plasma standards.    -   b. PK of rFVIIIFc and ADVATE®    -   c. PK properties of rFVIIIFc were compared with rFVIII (ADVATE®;        sequential PK subgroup). Overall study duration was ≤75 weeks        for all subjects. The primary efficacy endpoint was annualized        bleeding rate (ABR; Arms 1 and 2 vs. Arm 3). Prophylaxis dose        and interval, number of injections required for treatment of        bleeding episodes, and perioperative haemostasis were evaluated.

Key safety outcome measures included:

-   -   a. Incidence of inhibitor development. The study was powered to        detect the occurrence of inhibitors with a 2-sided 95%        confidence interval using the Clopper-Pearson exact method if 2        cases of inhibitor formation was observed.    -   b. Incidence of adverse events (AEs) occurring outside of the        perioperative management period

Adherence with treatment (adherence measures) was assessed using patientelectronic diaries.

A-LONG Results

Subjects

A total of 165 subjects were enrolled in the study. Median age was 30years (range, 12-65) and 8% were <18 years. The number of patients ineach arm of the study were: Arm 1 (individualized prophylaxis), n=118;Arm 2 (weekly prophylaxis), n=24; Arm 3 (episodic treatment), n=23; andperioperative management subgroup, n=9, 9 surgeries (8 subjects from Arm1 and 1 from Arm 2). Subjects from each treatment arm were eligible toenter the surgery subgroup if they required major surgery, had ≥12 EDsto rFVIIIFc and a negative inhibitor titre following this period andwithin 4 weeks prior to surgery. Patients on prior episodic treatmenthad a higher median number of bleeding episodes in the 12 months priorto the study, and a higher proportion of patients in Arms 2 and 3 hadtarget joints. For patients on prior prophylaxis, 87% reported injectingat least three times weekly.

92.7% of subjects completed the study. In total, 153 (approximately 93%)subjects completed the study; 112/118 (95%) in Arm 1, 19/24 (795) in Arm2, and 22/23 (96%) in Arm 3. Reasons for premature discontinuation were:Arm 1—subject withdrawal (n=2), physician decision (n=2), other (n=1),death (n=1); Arm 2—subject withdrawal (n=2; adverse events (AEs) relatedto study drug can have contributed), AEs (n=2), other (n=1); Arm 3—other(n=1).

Age, race, and geography of subjects were representative of the globalhaemophilia A population who have access to treatment, as summarized inTABLE 6.

TABLE 6 A-LONG subjects demographics Arm 1 Arm 2 Arm 3 Total Demographic(N = 118) (N = 24) (N = 23) N = 165 Age (years), median (min-max) 29.0(12-65)  31.5 (18-59)  34.0 (13-62)  30.0 (12-65)  Weight (kg), median(min-max)    71.65 (42.0-127.4)    75.85 (50.0-105.0)    70.00(48.0-110.4)    71.60 (42.0-127.4) BMI (kg/m²) median (min-max)  23.90(15.3-37.1)  24.60 (18.8-37.4)  22.80 (17.2-35.6)  23.90 (15.3-37.4)Race, n (%) White 79 (66.9) 12 (50.0) 16 (69.6) 107 (64.8)  Black 7(5.9) 1 (4.2) 2 (8.7) 10 (6.1)  Asian 27 (22.9) 11 (45.8)  5 (21.7) 43(26.1) Other 5 (4.2) 0 0 5 (3.0) Geographic location - n (%) Europe 34(28.8)  3 (12.5)  4 (17.4) 41 (24.8) North America 44 (37.3)  5 (20.8) 7 (30.4) 56 (33.9) Other* 40 (33.9) 16 (66.7) 12 (52.2) 68 (41.2)Genotype - n (%) Intron 22 inversion 41 (35.0)  7 (33.3)  9 (39.1) 57(35.4) Frameshift 24 (20.5)  4 (19.0)  6 (26.1) 34 (21.1) Missensemutation 22 (18.8)  4 (19.0) 1 (4.3) 27 (16.8) Nonsense mutation 19(16.2)  6 (28.6) 1 (4.3) 26 (16.1) Splice site change 7 (6.0) 0  4(17.4)11 (6.8)  Intron 1 inversion 3 (2.6) 0 1 (4.3) 4 (2.5) Duplication 1(0.9) 0 0 1 (0.6) NA 0 0 1 (4.3) 1 (0.6) von Willebrand factor antigen -118.0 (85, 151) 129.0 (86, 166) 131.0 (83, 155) 118.0 (85, 153) medianIU/dl (IQR) Pre-study FVIII regimen - n (%) Prophylaxis 87 (73.7) 0 0 87(52.7) Episodic 31 (26.3) 24 (100)  23 (100)  78 (47.3) Estimated no. ofbleeds prior 12 mo - median (IQR) † Prior prophylaxis 6.0 (2, 15) — —  6(2, 15) Prior episodic 27.0 (17, 41) 29.5 (19, 44) 24.0 (15, 36)  27(18, 40) ≥1 Target joint - n (%) Prior prophylaxis 47 (39.8) — — 47(28.5) Prior episodic 26 (22.0) 22 (91.7) 18 (78.3) 66 (40.0) Familyhistory of inhibitor 4 (3.4) 1 (4.2) 2 (8.7) 7 (4.2) HIV positive 25(21.2)  4 (16.7)  7 (30.4) 36 (21.8) HCV positive 55 (46.6) 14 (58.3) 13(56.5) 82 (49.7) BMI, body mass index; HIV, human immunodeficiencyvirus; HCV, hepatitis C virus IU/dl denotes international units perdeciliter, BMI body mass index, HIV human immunodeficiency virus, HCVhepatitis C virus, NA not applicable. *Other included Australia, NewZealand, Brazil, Hong Kong, India. Japan, Russia, and South Africa. †Calculation was based on available data.

Subjects received rFVIIFc for a median (min-max) of 30.5 (<1-54) weekswith a median (minimum to maximum) in arms 1, 2, and 3 of 32.1 (9, 54),28.0 (<1, 38), and 28.9 (15, 32) weeks, respectively. In total, 111subjects (67.7%) had >50 EDs to study drug. A total of 9356 injectionswere administered during the study, corresponding to 9170 EDs (100.2patient-years of exposure). Overall, 93.6% of patients were compliantwith both the prescribed dose and interval in the prophylaxis arms.

Overall, 93.6% of subjects were adherent with both the prescribed doseand the prescribed dosing interval in the prophylaxis treatment arms.

Efficacy

In total, 163 subjects were included in efficacy analyses. One subjectwas excluded due to only receiving ADVATE® (Arm 1); another subjectwithdrew before efficacy assessment (Arm 2). Median ABR with the 25thand 75th percentiles (interquartile range [IQR]) were as follows:Arm1—individualized prophylaxis arm: 1.6 (0.0, 4.7); Arm 2—weeklyprophylaxis arm: 3.6 (1.9, 8.4); and Arm 3—episodic treatment arm: 33.6(21.1, 48.7). Most bleeding episodes were spontaneous. In Arms 1, 2, and3, respectively, 45.3%, 17.4%, and 0% had no bleeding episodes and13.7%, 34.8%, and 0% had 1 bleeding episode.

Median Dosing Interval:

-   -   a. In the individualized prophylaxis arm, the median dosing        interval was 3.5 days, during the last 3 months on study.    -   b. 30 percent of patients in the individualized prophylaxis arm        achieved a mean dosing interval of at least 5 days.    -   c. Overall, the median (IQR) dosing interval with individualised        prophylaxis aimed to achieve a FVIII trough of at least 1 to 3        IU/dL (with the maximum 5-day dosing interval permitted in the        protocol) was 3.5 (3.2-4.4) days (Table 7) based on the median        (IQR) weekly dose of 78 (72-91) IU/kg.    -   d. Approximately 30% of subjects achieved a mean dosing interval        of 5 days over the last 3 months on study. 100% of subjects had        mean dosing intervals 2 days throughout the study.

The median dose per injection for Arm 3 episodic regimen was 26.5 IU/kg(n=23) and the median total dose per bleeding episode was 27.4 IU/kg(n=23).

TABLE 7 Comparative pharmacokinetics for rFVIIIFc vs. rFVIII (n = 28)¹Geometric mean of Geometric Geometric intra-subject mean for mean forratio rFVIIIFc PK rFVIII PK (95% CI) PK parameter (95% CI) (95% CI)p-value Elimination t_(1/2) (h) 18.97  12.43  1.53 (17.03, 21.12)(11.14, 13.86) (1.36, 1.71) <0.001 CL (mL/h/kg) 1.95 3.041 0.64 (1.71,2.22) (2.71, 3.41) (0.60, 0.69) <0.001 Time to 1 4.92 3.30  1.49 IU/dL(days) (4.43, 5.46) (2.99, 3.65) (1.41, 1.57) (50 IU/kg dose) <0.001¹Arm 1 sequential PK group, compartmental model, one-stage assay.Estimates and 95% CIs for geometric means and geometric mean ratios;statistical significance assessed at the 2-sided 0.05 level; PK,pharmacokinetics; CI, confidence interval; Cmax, maximal concentration;AUC, area-under-the-curve; t½, half-life; CL, clearance; MRT, meanresidence time; Vss, volume of distribution at steady state.

Control of bleeding: approximately 98% (97.7%) of bleeding episodes werecontrolled by one or two injections of rFVIIIFc. 87.3% of bleeds werecontrolled with one injection. 1.7% required three injections.

Perioperative management: Overall, 9 major surgeries were performed in 9subjects (8 subjects from Arm 1; 1 subject from Arm 2), including kneearthroplasty (n=5), laparoscopic inguinal hernia repair (n=2),appendectomy (n=1), and arthroscopy (n=1). Treating physicians rated thehemostatic efficacy of rFVIIIFc as excellent (8/9) or good (1/9) in 100%of surgeries. Median (min, max) estimated blood loss available for 7/9surgeries was 15.0 (0, 600) mL during surgery and 0.0 (0, 1100) mLpost-operatively; post-surgical drainage).

PK

Comparative PK data for rFVIIIFc and ADVATE® available from 28 subjectsin the Arm 1 sequential PK subgroup are summarised in Table 7.

-   -   a. rFVIIIFc demonstrated an approximate 50% longer elimination        half-life (FIG. 2) and mean residence time compared with ADVATE®        (P<0.001) due to a 35% reduction in rFVIIIFc clearance with        respect to ADVATE®.    -   b. Geometric mean time to 1 IU/dL FVIII activity following a 50        IU/kg dose was approximately 5 days for rFVIIIFc versus        approximately 3 days for ADVATE® (P<0.001; Table 7, FIG. 2).    -   c. Incremental recovery for rFVIIIFc was clinically comparable        with ADVATE®.    -   d. There was no shift in the PK properties of rFVIIIFc as        evidenced by comparable PK properties between the baseline and        repeat rFVIIIFc PK profiles.    -   e. Analogous PK results were obtained when the analysis was        based on the chromogenic FVIII assay.    -   f. The one-stage and chromogenic clotting assays accurately and        precisely measured both rFVIIIFc and ADVATE® utilising        commercially available plasma FVIII standards.

The geometric mean (95% confidence interval) terminal half-life ofrFVIIIFc was approximately 19.0 hours (17.0, 21.1) hours, which is1.53-fold longer than that of ADVATE® (approximately 12.4 (11.1, 13.9)hours).

Geometric mean time (IQR) to 1% FVIII activity following 50 IU/kg ofrFVIIIFc was approximately 5 days (4.92 (4.43, 5.46) days). ComparablePK profiles of rFVIIIFc were observed at Week 14. The median dosinginterval with individualized prophylaxis was 3.5 days and the mediandose per week was 78 IU/kg; approximately 30% of subjects achieved amean dosing interval of ≥5 days over the last 3 months on study(subjects with 26 months on study) as shown in Table 8. 98% of bleedingepisodes were controlled with 1-2 injections.

TABLE 8 Prophylactic dosing summary Arm 1 Arm 2 Individualised Weekly nprophylaxis n prophylaxis Median dose, IU/ kg/week (IQR) Overall 11777.9 23 65.6  (72.3, 91.2) (64.2, 68.2) Last 3 months¹ 112 77.7 16 65.5² (71.9, 106.2) (64.3, 67.3) Median dosing interval, days (IQR) Overall117  3.5 (3.2, 4.4) Last 3 months¹ 112  3.5³ (3.0, 5.0) ¹Based on last 3months on study for subjects on study ≥6 months ²Fixed dose of 65IU/kg/wk. ³30% of subjects achieved every 5-day pharmacokinetic-drivendosing to maintain troughs 1% to 3% above baseline. IQR, interquartilerange.

Safety

No inhibitors were detected to rFVIIIFc, and no cases of anaphylaxis,allergy, or serious thrombic events were reported.

rFVIIFc was generally well tolerated, and no serious adverse events wereassessed to be related to rFVIIIFc.

The most common AEs, regardless of causality, (incidence 25%) occurringoutside of the perioperative management period were nasopharyngitis,arthralgia (joint pain), headache, and upper respiratory tractinfection, as summarized in Table 9.

12 subjects (7.3%) experienced at least one serious AE (SAE) outside ofthe perioperative management period.

No SAEs were assessed to be related to drug by the investigator.

Insignificant incidence of non-neutralizing antibody (NNA) has beenobserved under NNA assay, which is approximately 20 times more sensitivethan Bethesda assay. Five subjects were found positive for NNAs atbaseline, but all had at least one negative evaluation during the study.Six subjects, four in Arm 1 study and two in Arm 2 study, becamepositive during the study. In almost all cases, however, antibodies weretransient and of low titer, and in all cases antibodies were directedagainst FVIII.

Overall, 14 (8.5%) subjects reported SAEs. No SAEs were determined bythe investigator as related to study drug, and SAEs reported were notexperienced in more than 1 subject. There was 1 death during the study,attributed to polysubstance overdose, and assessed as unrelated torFVIIIFc by the investigator.

TABLE 9 Summary of adverse events (AEs) Total N = 164 n (%) Any AE 108(65.9)  Most common AEs (≥5% of subjects) Nasopharyngitis 20 (12.2)Arthralgia 13 (7.9)  Headache 9 (5.5) Upper respiratory infection 9(5.5)

Summary

Individualized and weekly prophylactic regimens resulted in lowsingle-digit median annualized bleeding rates

In the individualized prophylaxis arm, the median dosing interval was3.5 days. During the last 3 months on study, 30 percent of patients inthe individualized prophylaxis arm achieved a mean dosing interval of atleast 5 days.

98% of bleeding episodes were controlled by one or two injections ofrFVIIIFc. In total, 757 bleeding episodes (Arm 1=209; Arm 2=92; Arm3=456) were treated in 106 of the 164 subjects. 87.3% required a singleinjection of rFVIIIFc for resolution, and 97.8% required ≤2 injections.A total of 85.6%, 80.4% and 89.5% of bleeding episodes were resolvedwith 1 injection of rFVIIIFc in Arms 1, 2 and 3, respectively.

Hemostatic efficacy of rFVIIIFc during surgery was rated by treatingphysicians as excellent or good in 100% of surgeries.

The half-life of rFVIIIFc was approximately 19.0 hours compared to 12.4hours for ADVATE®.

No subject developed an inhibitor or experienced an anaphylacticreaction to rFVIIIFc.

Recombinant FVIIIFc was generally well tolerated.

A-LONG was the largest registrational global pivotal phase 3 study oflong-lasting rFVIII in severe haemophilia A conducted to date. The studyshowed that rFVIIIFc offers the potential for a markedly reducedinjection frequency, decreased treatment burden, an improvement inclinical outcomes for prevention of bleeds in patients with severehaemophilia A.

Goal of the study design: The study was designed to evaluate theefficacy and safety of rFVIIIFc in the control and prevention ofbleeding episodes, routine prophylaxis, and perioperative management insubjects with severe hemophilia A. In addition, the study was designedto assess the effective dose and interval of rFVIIIFc for prophylaxis,as well as the feasibility of weekly treatment at a dose of 65 IU/Kg.

Episodic treatment is the administration of replacement factor only asneeded to treat bleeding episodes after they have started. Prophylactictreatment is the regular administration of replacement factor to preventbleeding episodes.

The starting regimen was 25 LU/kg on Day land 50 IU/kg on Day 4. Thedose and frequency of treatment could be adjusted based on the subject'sPK profile and the goal was to maintain trough factor levels sufficientto prevent bleeding episodes using doses between 25 IU/kg and 65 IU/kgand treatment intervals of 3 to 5 days.

More than half of severe hemophilia A patients in the US still do notfollow a prophylaxis regimen, due in part to the treatment burden. Wetherefore included the weekly dosing regimen in the A-LONG study toevaluate whether this regimen would result in a lower ABR compared to anepisodic regimen. The median ABR in the weekly regimen was 3.59 (IQR1.9, 8.4) compared with 33.6 (IQR 21.1, 48.7) for episodic treatment.

All subjects had an initial PK evaluation to characterize the PK ofrFVIIIFc in a representative population of patients with Hemophilia A.

More extensive PK sampling was conducted in a subset of subjects in theindividualized prophylaxis arm (Arm 1) at baseline after a single doseof ADVATE® (50 IU/kg), and after a 4 day washout, followed by a singledose of rFVIIIFc (50 IU/kg). Blood samples were taken for ADVATE® over aperiod of 72 hours. Blood samples were then taken for rFVIIIFc over aperiod of 120 hours. PK assessment of rFVIIIFc in this subset wasrepeated at 12 to 24 weeks with the same PK sampling schedule.

In peri-operative management, a single injection of rFVIIIFc wassufficient to maintain haemostasis to the end date/time of all majorsurgeries at a median dose of 51.4 IU/kg. Median rFVIIIFc consumption(summarized over all injections during each referenced time period) was80.6 IU/kg on the day of major surgery, 161.3 IU/kg for Days 1-3 daysfollowing surgery, and 387.1 IU/kg for Days 4-14 following surgery.Perioperative haemostasis with rFVIIIFc was rated as excellent or goodfor all 9 major surgeries. No subjects reported a bleeding episodeduring the postoperative or rehabilitation periods. Overall, 7 adverseevents (AEs) were reported in 4 (44.4%) subjects in the surgerysubgroup, of which 6 AEs were of mild or moderate severity, and 1 AE wasconsidered severe. Two serious AEs (inguinal hernia and appendicitis)were reported in 2 subjects. All AEs during the perioperative periodwere assessed by the investigators as unrelated to rFVIIIFc treatment.

Treating physicians rated the hemostatic efficacy of rFVIIIFc asexcellent or good in 100% of surgeries on a 4 point scale includingexcellent, good, fair, and poor/none.

rFVIIFc resulted in low median ABRs of 3.59 in the weekly prophylaxisarm and 1.60 in the individualized interval prophylaxis arm. Incontrast, the episodic treatment arm had a median ABR of 33.57.

In the individualized prophylaxis arm, the median dosing interval was3.5 days at a median dose of 77.7 FU/kg during the last 3 months onstudy.

The terminal half-life was approximately 18.97 hours for rFVIIIFc andapproximately 12.3 hours for ADVATE®.

97.8% of bleeding episodes were controlled by one or two injections.

Hemostatic efficacy of rFVIIIFc for perioperative management was ratedby treating physicians as excellent or good in 100% of surgeries.

Arm 2 was a weekly dosing regimen. It was designed to investigatebenefit for patients on prophylaxis therapy, and to compare thoseresults with subjects using an episodic treatment regimen. The medianABR in Arm 2 was 3.59 compared with 33.57 for the episodic treatmentregimen (Arm 3).

The median dose per injection required for resolution of bleeding was27.4 IU/kg, and the median total dose required was 28.2 IU/kg.

The median number of injections required for resolution of a bleedingepisode was consistently 1.0 when treatment was administered within 8hours of bleed onset, regardless of type or location of bleed. Overall,78.8% of rFVIIIFc injections in Arm 1, 64.8% in Arm 2, and 79.7% ofinjections in Arm 3, were rated by subjects as producing excellent orgood response. The median dosing interval of 3.5 days from Arm 1represents the dosing interval that can be achieved by the majority ofpatients: 30% of patients were able to achieve a dosing interval of atleast 5 days. Some patients were able to achieve a weekly prophylaxisregimen (Arm 2) with a median ABR of 3.59 bleeding episodes.

Subjects in the individualized prophylaxis arm received an initial doseof 25 IU/kg on Day 1 and 50 IU/kg on Day 4. The study design allowed foradjustment of the dosing interval and dose to maintain targeted troughfactor levels and to prevent bleeding. We believe the median dosinginterval and dose based on the last 6 months on study is mostrepresentative of the individualized prophylaxis regimen.

87.3% of bleeding episodes were resolved with one injection of rFVIIIFcand 97.8% were resolved with one or two injections. For the 96 (12.7%)bleeding episodes that required more than 1 injection for resolution,the median interval between the first and second injection was 30.9hours.

The investigators' global assessment of subject response to theirassigned rFVIIIFc regimen was rated as excellent or effective for 99.4%,100%, and 98.1% of the subject visits in Arms 1, 2, and 3, respectively.

In A-LONG, the clotting factor activity was measured using a one-stage(aPTT) clotting assay and a chromogenic assay. The reported half-livesare based on the results of the one-stage clotting assay. While thehalf-lives of rFVIIIFc and ADVATE® were both slightly longer with thechromogenic assay, the ratios between products were consistent betweenthe two assays.

EMBODIMENTS

E1. A pharmaceutical composition comprising:

-   -   (a) a FVIII polypeptide;    -   (b) one or more stabilizing agents selected from the group        consisting of sucrose, trehalose, raffinose, arginine, and        mixture thereof;    -   (c) sodium chloride (NaCl);    -   (d) L-histidine;    -   (e) calcium chloride; and    -   (f) polysorbate 20 or polysorbate 80.

E2. The pharmaceutical composition of embodiment E1, wherein mannitol,glycine, alanine, or hydroxyethyl starch is not included.

E3. The pharmaceutical composition of embodiment E1 or E2, wherein NaClis the only bulking agent.

E4. The pharmaceutical composition of any one of embodiments E1 to E3,comprising sucrose.

E5. The pharmaceutical composition of embodiment E4, comprising about 1%(w/v) to about 2.5% (w/v) sucrose.

E6. The pharmaceutical composition of embodiment E5, comprising about1.3% (w/v) sucrose to about 2.0% (w/v) sucrose.

E7. The pharmaceutical composition of embodiment E6, comprising about1.33% (w/v) sucrose or about 2.0% (w/v) sucrose.

E8. The pharmaceutical composition of embodiment E4, comprising about 10mg/ml to about 25 mg/ml sucrose.

E9. The pharmaceutical composition of embodiment E8, comprising about 13mg/ml to about 20 mg/ml sucrose.

E10. The pharmaceutical composition of embodiment E9, comprising about13.3 mg/ml sucrose or about 20.0 mg/ml sucrose.

E11. The pharmaceutical composition of any one of embodiments E1 to E10,comprising about 150 mM to about 250 mM NaCl.

E12. The pharmaceutical composition of embodiment E11, comprising about175 mM to about 225 mM NaCl.

E13. The pharmaceutical composition of embodiment E12, comprising about200 mM to about 210 mM NaCl.

E14. The pharmaceutical composition of embodiment E13, comprising about205 mM NaCl.

E15. The pharmaceutical composition of any one of embodiments E1 to E10,comprising about 8.8 mg/ml to about 14.6 mg/ml NaCl.

E16. The pharmaceutical composition of embodiment E15, comprising about10 mg/ml to about 13 mg/ml NaCl.

E17. The pharmaceutical composition of embodiment E16, comprising about12.0 mg/ml NaCl.

E18. The pharmaceutical composition of any one of embodiments E1 to E10,comprising about 250 mM to about 350 mM NaCl.

E19. The pharmaceutical composition of embodiment E18, comprising about275 mM to about 325 mM NaCl.

E20. The pharmaceutical composition of embodiment E19, comprising about308 mM NaCl.

E21. The pharmaceutical composition of any one of embodiments E1 to E10,comprising s about 14.6 mg/ml to about 20.5 mg/ml NaCl.

E22. The pharmaceutical composition of embodiment E21, comprising about16 mg/mi to about 19 mg/ml NaCl.

E23. The pharmaceutical composition of embodiment E22, comprising about18.0 mg/ml NaCl.

E24. The pharmaceutical composition of any one of embodiments E1 to E23,comprising about 5 mM to about 15 mM L-histidine.

E25. The pharmaceutical composition of embodiment E24, comprising about6.64 mM L-histidine or about 9.8 mM L-histidine.

E26. The pharmaceutical composition of any one of embodiments E1 to E23,comprising about 0.75 mg/ml to about 2.25 mg/ml L-histidine.

E27. The pharmaceutical composition of embodiment E26, thepharmaceutical composition comprises about 1.03 mg/ml L-histidine orabout 1.55 mg/ml L-histidine.

E28. The pharmaceutical composition of any one of embodiments E1 to E27,comprising about 5 mM to about 10 mM calcium chloride.

E29. The pharmaceutical composition of embodiment E28, comprising about5.4 mM calcium chloride or about 8 mM calcium chloride.

E30. The pharmaceutical composition of any one of embodiments E1 to E27,comprising about 0.75 mg/ml to about 1.5 mg/ml calcium chloridedihydrate.

E31. The pharmaceutical composition of embodiment E30, comprising about0.8 mg/ml calcium chloride dihydrate or about 1.18 mg/ml calciumchloride dihydrate.

E32. The pharmaceutical composition of any one of embodiments E1 to E31,comprising about 0.008% (w/v) to about 0.025% (w/v) polysorbate 20 orpolysorbate 80.

E33. The pharmaceutical composition of embodiment E32, comprising about0.013% (w/v) polysorbate 20 or polysorbate 80 or about 0.02% (w/v)polysorbate 20 or polysorbate 80.

E34. The pharmaceutical composition of any one of embodiments E1 to E31,comprising about 0.08 mg/ml to about 0.25 mg/ml polysorbate 20 orpolysorbate 80.

E35. The pharmaceutical composition of embodiment E34, comprising about0.13% mg/ml polysorbate 20 or polysorbate 80 or about 0.20 mg/mlpolysorbate 20 or polysorbate 80.

E36. The pharmaceutical composition of any one of embodiments E1 to E35,wherein the rFVIIIFc polypeptide comprises a first subunit comprising anamino acid sequence at least 90% or 95% identical to amino acids 20 to1684 of SEQ ID NO:2 or 20 to 2578 of SEQ ID NO:6, and a second subunitcomprising an amino acid sequence at least 90% to 95% identical to aminoacids 21 to 247 of SEQ ID NO:4.

E37. The pharmaceutical composition of embodiment E36, wherein therFVIIIFc polypeptide comprises a first subunit comprising amino acids 20to 1684 of SEQ ID NO:2 or 20 to 2578 of SEQ ID NO:6, and a secondsubunit comprising amino acids 21 to 247 of SEQ ID NO:4.

E38. The pharmaceutical composition of any one of embodiments E1 to E37,wherein the FVIII polypeptide is present at a concentration of about 50IU/ml to about 1500 IU/ml.

E39. The pharmaceutical composition of embodiment E38, comprising 83IU/ml, 167 IU/ml, 250 IU/ml, 333 IU/ml, 500 IU/ml, 667 IU/ml, or 1000IU/ml of the FVIII polypeptide.

E40. The pharmaceutical composition of any one of embodiments E1 to E37,wherein the FVIII polypeptide is present at a concentration of about 100IU/ml to about 2500 IU/ml.

E41. The pharmaceutical composition of embodiment E40, comprising 150IU/ml, 287.5 IU/ml, 431.25 IU/ml, 575 IU/ml, 862.5 IU/ml, 1150 IU/ml, or1725 IU/ml of the FVIII polypeptide.

E42. The pharmaceutical composition of any one of embodiments E1 to E4,comprising:

-   -   (a) about 50 IU/ml to about 1500 IU/ml of the FVIII polypeptide;    -   (b) about 1% (w/v) to about 2.5% (w/v) of sucrose;    -   (c) about 150 mM to about 250 mM NaCl;    -   (d) about 5 mM to about 15 mM L-histidine;    -   (e) about 5 mM to about 10 mM calcium chloride; and    -   (f) about 0.008% (w/v) to about 0.025% of polysorbate 20 or        polysorbate 80.

E43. The pharmaceutical composition of embodiment E42, comprising about175 mM to about 225 mM NaCl.

E44. The pharmaceutical composition of embodiment E43, comprising about200 mM to about 210 mM NaCl.

E45. The pharmaceutical composition of embodiment E44, comprising:

-   -   (a) about 83 IU/m, about 167 IU/ml, about 250 IU/mi, about 333        IU/ml, about 500 IU/ml, about 667 IU/ml, or about 1000 IU/ml of        the FVIII polypeptide;    -   (b) about 1.33% (w/v) of sucrose;    -   (c) about 205 mM NaCl;    -   (d) about 6.64 mM L-histidine;    -   (e) about 5.4 mM calcium chloride; and    -   (f) about 0.013% (w/v) of polysorbate 20 or polysorbate 80.

E46. The pharmaceutical composition of any one of embodiments E1 to E4,comprising:

-   -   (a) about 100 IU/ml to about 2500 IU/ml of the FVIII        polypeptide;    -   (b) about 1% (w/v) to about 2.5% (w/v) of sucrose;    -   (c) about 250 mM to about 350 mM NaCl;    -   (d) about 5 mM to about 15 mM L-histidine;    -   (e) about 5 mM to about 10 mM calcium chloride; and    -   (f) about 0.008% (w/v) to about 0.025% of polysorbate 20 or        polysorbate 80.

E47. The pharmaceutical composition of embodiment E46, comprising about275 mM to about 325 mM NaCl.

E48. The pharmaceutical composition of embodiment E47, comprising:

-   -   (a) about 150 IU/ml, about 287.5 IU/ml, about 431.25 IU/ml,        about 575 IU/ml, about 862.5 IU/ml, about 1150 IU/ml, or about        1725 IU/ml of the g FVIII polypeptide;    -   (b) about 2.0% (w/v) of sucrose;    -   (c) about 308 mM NaCl;    -   (d) about 9.8 mM L-histidine;    -   (e) about 8 mM calcium chloride; and    -   (f) about 0.020% (w/v) of polysorbate 20 or polysorbate 80.

E49. The pharmaceutical composition of any one of embodiments E1 to E4,comprising:

-   -   (a) about 50 IU/ml to about 1500 U/ml of the FVIII polypeptide;    -   (b) about 10 mg/ml to about 25 mg/ml of sucrose;    -   (c) about 8.8 mg/ml to about 14.6 mg/ml NaCl;    -   (d) about 0.75 mg/ml to about 2.25 mg/ml L-histidine;    -   (e) about 0.75 mg/ml to about 1.5 mg/ml calcium chloride        dihydrate; and    -   (f) about 0.08 mg/ml to about 0.25 mg/ml of polysorbate 20 or        polysorbate 80.

E50. The pharmaceutical composition of embodiment E49, comprising about10 mg/ml to 13 mg/ml NaCl.

E51. The pharmaceutical composition of embodiment E50, comprising:

-   -   (a) about 83 IU/ml, about 167 IU/ml, about 250 IU/ml, about 333        IU/ml, about 500 IU/ml, about 667 IU/ml, or about 1000 IU/ml of        the FVIII polypeptide;    -   (b) about 13.3 mg/ml of sucrose;    -   (c) about 12.0 mg/ml NaCl;    -   (d) about 1.03 mg/ml L-histidine;    -   (e) about 0.8 mg/ml calcium chloride dihydrate; and    -   (f) about 0.13 mg/ml of polysorbate 20 or polysorbate 80.

E52. The pharmaceutical composition of any one of embodiments E1 to E4,comprising:

-   -   (a) about 100 IU/mi to about 2500 IU/ml of the FVIII        polypeptide;    -   (b) about 10 mg/ml to about 25 mg/ml of sucrose;    -   (c) about 14.6 mg/ml to about 20.5 mg/ml NaCl;    -   (d) about 0.75 mg/ml to about 2.25 mg/ml L-histidine;    -   (e) about 0.75 mg/ml to about 1.5 mg/ml calcium chloride        dihydrate; and    -   (f) about 0.08 mg/ml to about 0.25 mg/ml of polysorbate 20 or        polysorbate 80.

E53. The pharmaceutical composition of embodiment E52, comprising about16 mg/ml to about 19 mg/ml NaCl.

E54. The pharmaceutical composition of embodiment E53, comprising:

-   -   (a) about 150 IU/ml, about 287.5 IU/m, about 431.25 IU/ml, about        575 IU/m, about 862.5 IU/ml, about 1150 IU/ml, or about 1725        IU/ml of the FVIII polypeptide;    -   (b) about 20.0 mg/ml of sucrose;    -   (c) about 18.0 mg/ml NaCl;    -   (d) about 1.55 mg/ml L-histidine;    -   (e) about 1.18 mg/ml calcium chloride dihydrate; and    -   (f) about 0.20 mg/ml of polysorbate 20 or polysorbate 80.

E55. A pharmaceutical kit comprising:

-   -   (a) a first container comprising a lyophilized powder, where the        powder comprises        -   (i) the FVIII polypeptide,        -   (ii) one or more stabilizing agents selected from the group            consisting of sucrose, trehalose, raffinose, arginine, and            mixture thereof;        -   (iii) sodium chloride (NaCl);        -   (iv) L-histidine;        -   (v) calcium chloride; and        -   (vi) polysorbate 20 or polysorbate 80; and    -   (b) a second container comprising sterilided water for        injections to be combined with the lyophilized powder of the        first container.

E56. The pharmaceutical kit of embodiment E55, wherein mannitol,glycine, alanine, or hydroxyethyl starch is not included.

E57. The pharmaceutical kit of embodiment E55 or E56, wherein NaCl isthe only bulking agent.

E58. The pharmaceutical kit of any one of embodiments E55 to E57,comprising:

-   -   (a) a first container comprising a lyophilized powder, where the        powder comprises        -   (i) about 250 IU, about 500 IU, about 750 IU, about 1000 IU,            about 1500 IU, about 2000 IU, or about 3000 IU of the FVIII            polypeptide,        -   (ii) about 40 mg of sucrose;        -   (iii) about 36 mg of sodium chloride;        -   (iv) about 3.1 mg of L-histidine;        -   (v) about 2.40 mg of calcium chloride dihydrate; and        -   (v) about 0.40 mg of polysorbate 20 or polysorbate 80; and    -   (b) a second container comprising sterilized water for        injections at a volume sufficient to produce, when combined with        the lyophilized powder of the first container, a solution        comprising:        -   (i) about 83 IU/ml, about 167 IU/ml, about 250 IU/ml, about            333 IU/ml, about 500 IU/ml, about 667 IU/ml, or about 1000            IU/ml of the FVIII polypeptide, respectively;        -   (ii) about 1.33% (w/v) of sucrose;        -   (iii) about 205 mM NaCl;        -   (iv) about 6.64 mM L-histidine;        -   (v) about 5.4 mM of calcium chloride; and        -   (vi) about 0.013% (w/v) of polysorbate 20 or polysorbate 80.

E59. The pharmaceutical kit of any one of embodiments E55 to E57,comprising:

-   -   (a) a first container comprising a lyophilized powder, where the        powder comprises        -   (i) about 250 IU, about 500 IU, about 750 IU, about 1000 IU,            about 1500 IU, about 2000 IU, or about 3000 IU of the FVIII            polypeptide,        -   (ii) about 40 mg of sucrose;        -   (iii) about 36 mg of sodium chloride;        -   (iv) about 3.1 mg of L-histidine;        -   (v) about 2.40 mg of calcium chloride dihydrate; and        -   (v) about 0.40 mg of polysorbate 20 or polysorbate 80; and    -   (b) a second container comprising sterilized water for        injections at a volume sufficient to produce, when combined with        the lyophilized powder of the first container, a solution        comprising:        -   (i) about 83 IU/ml, about 167 IU/ml, about 250 IU/ml, about            333 IU/ml, about 500 IU/ml, about 667 IU/m, or about 1000            IU/mi of the FVIII polypeptide, respectively;        -   (ii) about 13.3 mg/ml of sucrose;        -   (iii) about 12.0 mg/ml of NaCl;        -   (iv) about 1.03 mg/ml of L-histidine;        -   (v) about 0.80 mg/ml of calcium chloride; and        -   (vi) about 0.13 mg/ml of polysorbate 20 or polysorbate 80.

E60. The pharmaceutical kit of any one of embodiments E55 to E59,wherein the first container is a glass vial comprising a rubber stopper.

E61. The pharmaceutical kit of any one of embodiments E55 to E112,wherein the second container is a syringe body, and wherein the syringebody is associated with a plunger.

E62. The pharmaceutical kit of embodiment E61, further comprising anadaptor to connect the glass vial to the syringe body.

E63. The pharmaceutical kit of embodiment E61 or E62, further comprisinginfusion tubing associated with a needle to be connected to the syringe,suitable for intravenous infusion.

E64. A method of reducing the annualized bleeding rate in a subjecthaving hemophilia comprising administering to the subject thepharmaceutical composition of any one of embodiments E1 to E54 at adosing interval of about three days or longer.

E65. The method of embodiment E64, wherein the administration isprophylactic and individualized for the subject resulting in anannualized bleeding rate less than about 5.0, less than about 4.9, lessthan about 4.8, less than about 4.7, less than about 4.6, or less thanabout 4.5.

E66. The method of embodiment E65, wherein the median annualizedbleeding rate is about 1.6.

E67. The method of embodiment E64, wherein the administration isprophylactic and weekly resulting in an annualized bleeding rate lessthan about 9.0, less than about 8.9, less than about 8.8, less thanabout 8.7, less than about 8.6, less than about 8.5, or less than about8.4.

E68. The method of embodiment E67, wherein the median annualizedbleeding rate is about 3.6.

E69. The method of embodiment E64, wherein the administration ison-demand or episodic resulting in an annualized bleeding rate less thanabout 55, less than about 54, less than about 53, less than about 52,less than about 51, less than about 50, less than about 49, less thanabout 48, or less than about 47.

E70. The method of embodiment E69, wherein the median annualizedbleeding rate is about 33.6.

E71. The method of any one of embodiments E64 to E70, wherein theeffective dose is between about 20/IU/kg to about 90 IU/kg.

E72. The method of any one of embodiments E64 to E71, wherein theeffective dose is 20-30 RU/kg, 30-40 IU/kg, 40-50 IU/kg, 50-60 IU/kg,60-70 IU/kg, 70-80 IU/kg, or 80-90 IU/kg.

E73. The method of any one of embodiments E64 to E72, wherein theeffective dose is 20 IU/kg, 25 IU/kg, 30 IU/kg, 35 IU/kg, 40 IU/kg, 45IU/kg, 50 IU/kg, 55 IU/kg, 60 IU/kg, 65 IU/kg, 70 IU/kg, 75 IU/kg, 80IU/kg, 85 IU/kg, or 90 IU/kg.

E74. The method of any of embodiments E71 to E73, wherein theadministration is prophylactic and individualized at an effective doseof about 25 IU/kg to about 65 IU/kg twice weekly or every three days orabout 50 IU/kg to about 65 IU/kg every 4 or 5 days.

E75. The method of embodiment E74, wherein a first dose of the FVIIIpolypeptide is administered is about 25 IU/kg and a second dose of theFVIII polypeptide is about 50 IU/kg.

E76. The method of any of embodiments E71 to E73, wherein theadministration is prophylactic and weekly at an effective dose of 65IU/kg weekly.

E77. The method of any of embodiments E71 to E73, wherein theadministration is on-demand or episodic at an effective dose of 10 IU/kgto 75 IU/kg every 12 to 24 hours.

E78. The method of any one of embodiments E64 to E70, wherein theeffective dose is a fixed dose, which is standard across all bodyweights.

E79. The method of embodiment E78, wherein the fixed dose is about 2,000IU, about 2,500 IU, about 3,000 IU, about 3,500 IU, or about 4,000 IUper dose.

E80. The method of any one of embodiments E64 to E70, wherein theeffective dose is a stratified dose.

E81. The method of anyone of embodiments E64 to E70, wherein a troughlevel of the FVIII polypeptide is above 1%, above 2%, or above 3% ofnormal.

E82. The method of any one of embodiments E64 to E81, wherein the FVIIIpolypeptide has a T_(1/2 beta) (activity) of about 7 hours to about 48hours, about 6 hours to about 49 hours, about 5 hours to about 50 hours.

E83. The method of any one of embodiment E82, wherein the FVIII has aTia (activity) mean of at least about 15 hours, at least about 16 hours,at least about 17 hours, at least about 18 hours, at least about 19hours, at least about 20 hours, at least about 21 hours, at least about22 hours, at least about 23 hours, at least about 24 hours, at leastabout 25 hours.

E84. The method of embodiment E83, wherein the T_(1/2 beta) (activity)mean is about 19 hours.

E85. The method of embodiment E82 or E83, wherein the T_(1/2 beta)(activity) mean is at least about 1.5 fold higher than a polypeptideconsisting of amino acids 20 to 1457 of SEQ ID NO:2, 20 to 2351 of SEQID NO: 6, or ADVATE®.

E86. The method of any one of embodiments E64 to E85, wherein the plasmatrough level of the FVIII polypeptide is maintained between about 1% andabout 5%, between about 1% and about 6%, between about 1% and about 7%,between about 1% and about 8%, between about 1% and about 9%, betweenabout 1% and about 10%, between about 1% and about 11%, between about 1%and about 12%, between about 1% and about 13%, between about 1% andabout 14%, between about 1% and about 15% above the baseline in thesubject.

E87. The method of any one of embodiments E64 to E86, wherein the FVIIIpolypeptide is a long-acting FVIII polypeptide comprising a FVIIIpolypeptide and a heterologous moiety.

E88. The method of embodiment E87, wherein the heterologous moiety is anFcRn binding partner.

E89. The method of embodiment E88, wherein the FcRn binding partnercomprises an Fc region.

E90. The method of embodiment E88 or E89, wherein the long-acting FVIIIpolypeptide further comprises a second FcRn binding partner.

E91. The method of embodiment E90, wherein the second FcRn bindingpartner comprises a second Fc region.

E92. The method of embodiment E90 or E91, wherein the FcRn bindingpartner and the second FcRn binding partner are associated.

E93. The method of embodiment E92, wherein the association is a covalentbond.

E94. The method of embodiment E93, wherein the covalent bond is adisulfide bond.

E95. The method of any one of embodiments E90 to E94, wherein the secondFcRn binding partner is not linked to an amino acid sequence by apeptide bond.

E96. The method of any one of embodiments E64 to E95, wherein thelong-acting FVIII polypeptide is FVIII monomer dimer hybrid.

E97. The method of any one of embodiments E87 to E96, wherein the FVIIIpolypeptide in the long-acting polypeptide is a human FVIII.

E98. The method of any one of embodiments E87 to E97, wherein the FVIIIpolypeptide in the long-acting polypeptide is a full-length FVIII or aB-domain deleted FVIII.

E99. The method of any one of embodiments E87 to E97, wherein the FVIIIpolypeptide is single chain.

E100. The method of any one of embodiments E87 to E97, wherein the FVIIIpolypeptide is unprocessed.

E101. The method of any one of embodiments E87 to E97, wherein the FVIIIpolypeptide is in two chains, a first chain comprising a heavy chain ofthe FVIII polypeptide and a second chain comprising a light chain of theFVIII polypeptide.

E102. The method of any one of embodiments E88 to E101, wherein the FcRnbinding partner in the chimeric polypeptide is a human Fc.

E103. The method of any one of embodiments E87 to E102, wherein theFVIII polypeptide is at least 60%, 70%, 80%, 90%, 95%, 95%, 97%, 98%,99%, or 100% identical to a FVIII amino acid sequence shown in Table 11Aor 11B without a signal sequence (amino acids 20 to 1457 of SEQ ID NO: 2or amino acids 20 to 2351 of SEQ ID NO: 6).

E104. The method of any one of embodiments E88 to E103, wherein the FcRnbinding partner is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%,or 100% identical to a Fc amino acid sequence shown in Table 11B withouta signal sequence (amino acids 21 to 247 SEQ ID NO:4).

E105. The method of any one of embodiments E90 to E104, wherein thesecond FcRn binding partner in the chimeric polypeptide is a human Fc.

E106. The method of embodiment E105, wherein the second FcRn bindingpartner is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to a Fc amino acid sequence shown in Table 11B without asignal sequence (amino acids 21 to 247 SEQ ID NO:4)

E107. The method of any one of embodiments E64 to E106, wherein thefixed dose of the clotting factor is for perioperative management of ableeding episode.

E108. The method of anyone of embodiments E64 to E107, wherein thesubject is in need of controlling or preventing bleeding or bleedingepisodes.

E109. The method of embodiment E108, wherein the subject is in need ofcontrolling or preventing bleeding in minor hemorrhage, hemarthroses,superficial muscle hemorrhage, soft tissue hemorrhage, moderatehemorrhage, intramuscle or soft tissue hemorrhage with dissection,mucous membrane hemorrhage, hematuria, major hemorrhage, hemorrhage ofthe pharynx, hemorrhage of the retropharynx, hemorrhage of theretroperitonium, hemorrhage of the central nervous system, bruises,cuts, scrapes, joint hemorrhage, nose bleed, mouth bleed, gum bleed,intracranial bleeding, intraperitoneal bleeding, minor spontaneoushemorrhage, bleeding after major trauma, moderate skin bruising, orspontaneous hemorrhage into joints, muscles, internal organs or thebrain.

E110. The method of embodiment E109, wherein the subject is in need ofmanagement of bleeding associated with surgery or dental extraction.

E111. The method of embodiment E110, wherein the subject will undergo,is undergoing, or has undergone major surgery.

E112. The method of embodiment E111, wherein the major surgery isorthopedic surgery, extensive oral surgery, urologic surgery, or herniasurgery.

E113. The method of embodiment E112, wherein the orthopedic surgery isreplacement of knee, hip, or other major joint.

E114. The method of anyone of embodiments E64 to E113, wherein thesubject is in need of long-term treatment.

E115. The method of any one of embodiments E64 to E114, wherein theFVIII polypeptide is administered intravenously or subcutaneously.

E116. The pharmaceutical composition of any one of embodiments E18-E23,E40, E41, E46-E48, and E52-E54, wherein the pharmaceutical compositionis a pre-lyophilization composition.

E117. The pharmaceutical composition of any one of embodiments E1 toE37, wherein the FVIII polypeptide is present at a concentration ofabout 50 IU/ml to about 2500 IU/ml.

E118. The pharmaceutical composition of embodiment E117, comprising 1333IU/ml, 1667 IU/ml, or 2000 IU/ml of the FVIII polypeptide.

E119. The pharmaceutical composition of any one of embodiments E1 toE37, wherein the FVIII polypeptide is present at a concentration ofabout 100 IU/ml to about 4500 IU/ml.

E120. The pharmaceutical composition of embodiment E119, comprising 2300IU/mi, about 2875 IU/ml, or about 3450 IU/ml of the FVIII polypeptide.

E121. The pharmaceutical composition of any one of embodiments E1 to E4,comprising:

-   -   (a) about 50 IU/ml to about 2500 IU/ml of the FVIII polypeptide;    -   (b) about 1% (w/v) to about 2.5% (w/v) of sucrose;    -   (c) about 150 mM to about 250 mM NaCl;    -   (d) about 5 mM to about 15 mM L-histidine;    -   (e) about 5 mM to about 10 mM calcium chloride; and    -   (f) about 0.008% (w/v) to about 0.025% of polysorbate 20 or        polysorbate 80.

E122. The pharmaceutical composition of embodiment E121, comprisingabout 175 mM to about 225 mM NaCl.

E123. The pharmaceutical composition of embodiment E122, comprisingabout 200 mM to about 210 mM NaCl.

E124. The pharmaceutical composition of embodiment E123, comprising:

-   -   (a) about 1333 IU/ml, about 1667 IU/ml, or about 2000 IU/ml of        the FVIII polypeptide;    -   (b) about 1.33% (w/v) of sucrose;    -   (c) about 205 mM NaCl;    -   (d) about 6.64 mM L-histidine;    -   (e) about 5.4 mM calcium chloride; and    -   (f) about 0.013% (w/v) of polysorbate 20 or polysorbate 80.

E125. The pharmaceutical composition of any one of embodiments E1 to E4,comprising:

-   -   (a) about 100 IU/ml to about 4000 IU/ml of the FVIII        polypeptide;    -   (b) about 1% (w/v) to about 2.5% (w/v) of sucrose;    -   (c) about 250 mM to about 350 mM NaCl;    -   (d) about 5 mM to about 15 mM L-histidine;    -   (e) about 5 mM to about 10 mM calcium chloride; and    -   (f) about 0.008% (w/v) to about 0.025% of polysorbate 20 or        polysorbate 80.

E126. The pharmaceutical composition of embodiment E125, comprisingabout 275 mM to about 325 mM NaCl.

E127. The pharmaceutical composition of embodiment E126, comprising:

-   -   (a) about 2300 IU/ml, about 2875 IU/ml, or about 3450 IU/ml of        the g FVIII polypeptide;    -   (b) about 2.0% (w/v) of sucrose;    -   (c) about 308 mM NaCl;    -   (d) about 9.8 mM L-histidine;    -   (e) about 8 mM calcium chloride; and    -   (f) about 0.020% (w/v) of polysorbate 20 or polysorbate 80.

E128. The pharmaceutical composition of any one of embodiments E1 to E4,comprising:

-   -   (a) about 50 IU/ml to about 2500 IU/ml of the FVIII polypeptide;    -   (b) about 10 mg/ml to about 25 mg/ml of sucrose;    -   (c) about 8.8 mg/ml to about 14.6 mg/ml NaCl;    -   (d) about 0.75 mg/ml to about 2.25 mg/ml L-histidine;    -   (e) about 0.75 mg/ml to about 1.5 mg/ml calcium chloride        dihydrate; and    -   (f) about 0.08 mg/ml to about 0.25 mg/ml of polysorbate 20 or        polysorbate 80.

E129. The pharmaceutical composition of embodiment E128, comprisingabout 10 mg/ml to 13 mg/ml NaCl.

E130. The pharmaceutical composition of embodiment E129, comprising:

-   -   (a) about 1333 IU/m, about 1667 IU/ml, or about 2000 IU/ml of        the FVIII polypeptide;    -   (b) about 13.3 mg/ml of sucrose;    -   (c) about 12.0 mg/ml NaCl;    -   (d) about 1.03 mg/ml L-histidine;    -   (e) about 0.8 mg/ml calcium chloride dihydrate; and    -   (f) about 0.13 mg/ml of polysorbate 20 or polysorbate 80.

E131. The pharmaceutical composition of any one of embodiments E1 to E4,comprising:

-   -   (a) about 100 IU/ml to about 4000 IU/ml of the FVIII        polypeptide;    -   (b) about 10 mg/ml to about 25 mg/ml of sucrose;    -   (c) about 14.6 mg/ml to about 20.5 mg/ml NaCl;    -   (d) about 0.75 mg/ml to about 2.25 mg/ml L-histidine;    -   (e) about 0.75 mg/ml to about 1.5 mg/ml calcium chloride        dihydrate; and    -   (f) about 0.08 mg/ml to about 0.25 mg/ml of polysorbate 20 or        polysorbate 80.

E132. The pharmaceutical composition of embodiment E131, comprisingabout 16 mg/mi to about 19 mg/ml NaCl.

E133. The pharmaceutical composition of embodiment E132, comprising:

-   -   (a) about 2300 IU/ml, about 2875 IU/ml, or about 3450 IU/ml of        the FVIII polypeptide;    -   (b) about 20.0 mg/ml of sucrose,    -   (c) about 18.0 mg/ml NaCl;    -   (d) about 1.55 mg/ml L-histidine;    -   (e) about 1.18 mg/ml calcium chloride dihydrate; and    -   (f) about 0.20 mg/ml of polysorbate 20 or polysorbate 80.

E134. The pharmaceutical kit of any one of embodiments E55 to E57,comprising:

-   -   (a) a first container comprising a lyophilized powder, where the        powder comprises        -   (i) about 4000 IU, about 5000 IU, or about 6000 IU of the            FVIII polypeptide,        -   (ii) about 40 mg of sucrose;        -   (iii) about 36 mg of sodium chloride;        -   (iv) about 3.1 mg of L-histidine;        -   (v) about 2.40 mg of calcium chloride dihydrate; and        -   (v) about 0.40 mg of polysorbate 20 or polysorbate 80; and    -   (b) a second container comprising sterilized water for        injections at a volume sufficient to produce, when combined with        the lyophilized powder of the first container, a solution        comprising:        -   (i) about 1333 IU/ml, about 1667 IU/ml, or about 2000 IU/ml            of the FVIII polypeptide, respectively;        -   (ii) about 1.33% (w/v) of sucrose;        -   (iii) about 205 mM NaCl;        -   (iv) about 6.64 mM L-histidine;        -   (v) about 5.4 mM of calcium chloride; and        -   (vi) about 0.013% (w/v) of polysorbate 20 or polysorbate 80.

E135. The pharmaceutical kit of anyone of embodiments E55 to E57,comprising:

-   -   (a) a first container comprising a lyophilized powder, where the        powder comprises        -   (i) about 4000 IU, about 5000 IU, or about 6000 IU of the            FVIII polypeptide,        -   (ii) about 40 mg of sucrose;        -   (iii) about 36 mg of sodium chloride;        -   (iv) about 3.1 mg of L-histidine;        -   (v) about 2.40 mg of calcium chloride dihydrate; and        -   (v) about 0.40 mg of polysorbate 20 or polysorbate 80; and    -   (b) a second container comprising sterilized water for        injections at a volume sufficient to produce, when combined with        the lyophilized powder of the first container, a solution        comprising:        -   (i) about 1333 IU/ml, about 1667 IU/ml, or about 2000 IU/ml            of the FVIII polypeptide, respectively;        -   (ii) about 13.3 mg/ml of sucrose;        -   (iii) about 12.0 mg/ml of NaCl;        -   (iv) about 1.03 mg/ml of L-histidine;        -   (v) about 0.80 mg/ml of calcium chloride; and        -   (vi) about 0.13 mg/ml of polysorbate 20 or polysorbate 80.

E136. The pharmaceutical kit of anyone of embodiments E134 to E135,wherein the first container is a glass vial comprising a rubber stopper.

E137. The pharmaceutical kit of anyone of embodiments E134 to E136,wherein the second container is a syringe body, and wherein the syringebody is associated with a plunger.

E138. The pharmaceutical kit of embodiment E137, further comprising anadaptor to connect the glass vial to the syringe body.

E139. The pharmaceutical kit of embodiment E137 or E138, furthercomprising infusion tubing associated with a needle to be connected tothe syringe, suitable for intravenous infusion.

E140. A method of treating a bleeding disorder in a subject havinghemophilia comprising administering to the subject the pharmaceuticalcomposition of any one of embodiments E117 to E133 at a dosing intervalof about three days or longer.

E141. The method of embodiment E140, wherein the pharmaceuticalcomposition is administered for individual prophylaxis of hemophilia.

E142. The method of embodiment E140, wherein the pharmaceuticalcomposition is administered for weekly prophylaxis of hemophilia.

E143. The method of embodiment E140, wherein the pharmaceuticalcomposition is administered for episodic (on-demand) treatment ofhemophilia.

E144. The method of embodiment E140, wherein the pharmaceuticalcomposition is administered for perioperative management of hemophilia.

TABLE 10 Polynucleotide Sequences A. B-Domain Deleted FVIIIFc(i) B-Domain Deleted FVIIIFc Chain DNA Sequence (FVIII signalpeptide underlined, Fc region in bold) (SEQ ID NO: 1,which encodes SEQ ID NO: 2) 661                                A TGCAAATAGA GCTCTCCACC TGCTTCTTTC 721 TGTGCCTTTT GCGATTCTGC TTTAGTGCCA CCAGAAGATA CTACCTGGGT GCAGTGGAAC 781 TGTCATGGGA CTATATGCAA AGTGATCTCG GTGAGCTGCC TGTGGACGCA AGATTTCCTC 841 CTAGAGTGCC AAAATCTTTT CCATTCAACA CCTCAGTCGT GTACAAAAAG ACTCTGTTTG 901 TAGAATTCAC GGATCACCTT TTCAACATCG CTAAGCCAAG GCCACCCTGG ATGGGTCTGC 961 TAGGTCCTAC CATCCAGGCT GAGGTTTATG ATACAGTGGT CATTACACTT AAGAACATGG1021 CTTCCCATCC TGTCAGTCTT CATGCTGTTG GTGTATCCTA CTGGAAAGCT TCTGAGGGAG1081 CTGAATATGA TGATCAGACC AGTCAAAGGG AGAAAGAAGA TGATAAAGTC TTCCCTGGTG1141 GAAGCCATAC ATATGTCTGG CAGGTCCTGA AAGAGAATGG TCCAATGGCC TCTGACCCAC1201 TGTGCCTTAC CTACTCATAT CTTTCTCATG TGGACCTGGT AAAAGACTTG AATTCAGGCC1261 TCATTGGAGC CCTACTAGTA TGTAGAGAAG GGAGTCTGGC CAAGGAAAAG ACACAGACCT1321 TGCACAAATT TATACTACTT TTTGCTGTAT TTGATGAAGG GAAAAGTTGG CACTCAGAAA1381 CAAAGAACTC CTTGATGCAG GATAGGGATG CTGCATCTGC TCGGGCCTGG CCTAAAATGC1441 ACACAGTCAA TGGTTATGTA AACAGGTCTC TGCCAGGTCT GATTGGATGC CACAGGAAAT1501 CAGTCTATTG GCATGTGATT GGAATGGGCA CCACTCCTGA AGTGCACTCA ATATTCCTCG1561 AAGGTCACAC ATTTCTTGTG AGGAACCATC GCCAGGCGTC CTTGGAAATC TCGCCAATAA1621 CTTTCCTTAC TGCTCAAACA CTCTTGATGG ACCTTGGACA GTTTCTACTG TTTTGTCATA1681 TCTCTTCCCA CCAACATGAT GGCATGGAAG CTTATGTCAA AGTAGACAGC TGTCCAGAGG1741 AACCCCAACT ACGAATGAAA AATAATGAAG AAGCGGAAGA CTATGATGAT GATCTTACTG1801 ATTCTGAAAT GGATGTGGTC AGGTTTGATG ATGACAACTC 7CCTTCCTTT ATCCAAATTC1861 GCTCAGTTGC CAAGAAGCAT CCTAAAACTT GGGTACATTA CATTGCTGCT GAAGAGGAGG1921 ACTGGGACTA TGCTCCCTTA GTCCTCGCCC CCGATGACAG AAGTTATAAA AGTCAATATT1981 TGAACAATGG CCCTCAGCGG ATTGGTAGGA AGTACAAAAA AGTCCGATTT ATGGCATACA2041 CAGATGAAAC CTTTAAGACT CGTGAAGCTA TTCAGCATGA ATCAGGAATC TTGGGACCTT2101 TACTTTATGG GGAAGTTGGA GACACACTGT TGATTATATT TAAGAATCAA GCAAGCAGAC2161 CATATAACAT CTACCCTCAC GGAATCACTG ATGTCCGTCC TTTGTATTCA AGGAGATTAC2221 CAAAAGGTGT AAAACATTTG AAGGATTTTC CAATTCTGCC AGGAGAAATA TTCAAATATA2281 AATGGACAGT GACTGTAGAA GATGGGCCAA CTAAATCAGA TCCTCGGTGC CTGACCCGCT2341 ATTACTCTAG TTTCGTTAAT ATGGAGAGAG ATCTAGCTTC AGGACTCATT GGCCCTCTCC2401 TCATCTGCTA CAAAGAATCT GTAGATCAAA GAGGAAACCA GATAATGTCA GACAAGAGGA2461 ATGTCATCCT GTTTTCTGTA TTTGATGAGA ACCGAAGCTG GTACCTCACA GAGAATATAC2521 AACGCTTTCT CCCCAATCCA GCTGGAGTGC AGCTTGAGGA TCCAGAGTTC CAAGCCTCCA2581 ACATCATGCA CAGCATCAAT GGCTATGTTT TTGATAGTTT GCAGTTGTCA GTTTGTTTGC2641 ATGAGGTGGC ATACTGGTAC ATTCTAAGCA TTGGAGCACA GACTGACTTC CTTTCTGTCT2701 TCTTCTCTGG ATATACCTTC AAACACAAAA TGGTCTATGA AGACACACTC ACCCTATTCC2761 CATTCTCAGG AGAAACTGTC TTCATGTCGA TGGAAAACCC AGGTCTATGG ATTCTGGGGT2821 GCCACAACTC AGACTTTCGG AACAGAGGCA TGACCGCCTT ACTGAAGGTT TCTAGTTGTG2881 ACAAGAACAC TGGTGATTAT TACGAGGACA GTTATGAAGA TATTTCAGCA TACTTGCTGA2941 GTAAAAACAA TGCCATTGAA CCAAGAAGCT TCTCTCAAAA CCCACCAGTC TTGAAACGCC3001 ATCAACGGGA AATAACTCGT ACTACTCTTC AGTCAGATCA AGAGGAAATT GACTATGATG3061 ATACCATATC AGTTGAAATG AAGAAGGAAG ATTTTGACAT TTATGATGAG GATGAAAATC3121 AGAGCCCCCG CAGCTTTCAA AAGAAAACAC GACACTATTT TATTGCTGCA GTGGAGAGGC3181 TCTGGGA7TA TGGGATGAGT AGCTCCCCAC ATGTTCTAAG AAACAGGGCT CAGAGTGGCA3241 GTGTCCCTCA GTTCAAGAAA GTTGTTTTCC AGGAATTTAC TGATGGCTCC TTTACTCAGC3301 CCTTATACCG TGGAGAACTA AATGAACATT TGGGACTCCT GGGGCCATAT ATAAGAGCAG3361 AAGTTGAAGA TAATATCATG GTAACTTTCA GAAATCAGGC CTCTCGTCCC TATTCCTTCT3421 ATTCTAGCCT TATTTCTTAT GAGGAAGATC AGAGGCAAGG AGCAGAACCT AGAAAAAACT3481 TTGTCAAGCC TAATGAAACC AAAACTTACT TTTGGAAAGT GCAACATCAT ATGGCACCCA3541 CTAAAGATGA GTTTGACTGC AAAGCCTGGG CTTATTTCTC TGATGTTGAC CTGGAAAAAG3601 ATGTGCACTC AGGCCTGATT GGACCCCTTC TGGTCTGCCA CACTAACACA CTGAACCCTG3661 CTCATGGGAG ACAAGTGACA GTACAGGAAT TTGCTCTGTT TTTCACCATC TTTGATGAGA3721 CCAAAAGCTG GTACTTCACT GAAAATATGG AAAGAAACTG CAGGGCTCCC TGCAATATCC3781 AGATGGAAGA TCCCACTTTT AAAGAGAATT ATCGCTTCCA TGCAATCAAT GGCTACATAA3841 TGGATACACT ACCTGGCTTA GTAATGGCTC AGGATCAAAG GATTCGATGG TATCTGCTCA3901 GCATGGGCAG CAATGAAAAC ATCCATTCTA TTCATTTCAG TGGACATGTG TTCACTGTAC3961 GAAAAAAAGA GGAGTATAAA ATGGCACTGT ACAATCTCTA TCCAGGTGTT TTTGAGACAG4021 TGGAAATGTT ACCATCCAAA GCTGGAATTT GGCGGGTGGA ATGCCTTATT GGCGAGCATC4081 TACATGCTGG GATGAGCACA CTTTTTCTGG TGTACAGCAA TAAGTGTCAG ACTCCCCTGG4141 GAATGGCTTC TGGACACATT AGAGATTTTC AGATTACAGC TTCAGGACAA TATGGACAGT4201 GGGCCCCAAA GCTGGCCAGA CTTCATTATT CCGGATCAAT CAATGCCTGG AGCACCAAGG4261 AGCCCTTTTC TTGGATCAAG GTGGATCTGT TGGCACCAAT GATTATTCAC GGCATCAAGA4321 CCCAGGGTGC CCGTCAGAAG TTCTCCAGCC TCTACATCTC TCAGTTTATC ATCATGTATA4381 GTCTTGATGG GAAGAAGTGG CAGACTTATC GAGGAAATTC CACTGGAACC TTAATGGTCT4441 TCTTTGGCAA TGTGGATTCA TCTGGGATAA AACACAATAT TTTTAACCCT CCAATTATTG4501 CTCGATACAT CCGTTTGCAC CCAACTCATT ATAGCATTCG CAGCACTCTT CGCATGGAGT4561 TGATGGGCTG TGATTTAAAT AGTTGCAGCA TGCCATTGGG AATGGAGAGT AAAGCAATAT4621 CAGATGCACA GATTACTGCT TCATCCTACT TTAGCAATAT GTTTGCCACC TGGTCTCCTT4681 CAAAAGCTCG ACTTCACCTC CAAGGGAGGA GTAATGCCTG GAGACCTCAG GTGAATAATC4741 CAAAAGAGTG GCTGCAAGTG GACTTCCAGA AGACAATGAA AGTCACAGGA GTAACTACTC4801 AGGGAGTAAA ATCTCTGCTT ACCAGCATGT ATGTGAAGGA GTTCCTCATC TCCAGCAGTC4861 AAGATGGCCA TCAGTGGACT CTCTTTTTTC AGAATGGCAA AGTAAAGGTT TTTCAGGGAA4921 ATCAAGACTC CTTCACACCT GTGGTGAACT CTCTAGACCC ACCGTTACTG ACTCGCTACC4981 TTCGAATTCA CCCCCAGAGT TGGGTGCACC AGATTGCCCT GAGGATGGAG GTTCTGGGCT5041 GCGAGGCACA GGACCTCTAC GACAAAACTC ACACATGCCC ACCGTGCCCA GCTCCAGAAC5101 TCCTGGGCGG ACCGTCAGTC TTCCTCTTCC CCCCAAAACC CAAGGACACC CTCATGATCT5161 CCCGGACCCC TGAGGTCACA TGCGTGGTGG TGGACGTGAG CCACGAAGAC CCTGAGGTCA5221 AGTTCAACTG GTACGTGGAC GGCGTGGAGG TGCATAATGC CAAGACAAAG CCGCGGGAGG5281 AGCAGTACAA CAGCACGTAC CGTGTGGTCA GCGTCCTCAC CGTCCTGCAC CAGGACTGGC5341 TGAATGGCAA GGAGTACAAG TGCAAGGTCT CCAACAAAGC CCTCCCAGCC CCCATCGAGA5401 AAACCATCTC CAAAGCCAAA GGGCAGCCCC GAGAACCACA GGTGTACACC CTGCCCCCAT5461 CCCGGGATGA GCTGACCAAG AACCAGGTCA GCCTGACCTG CCTGGTCAAA GGCTTCTATC5521 CCAGCGACAT CGCCGTGGAG TGGGAGAGCA ATGGGCAGCC GGAGAACAAC TACAAGACCA5581 CGCCTCCCGT GTTGGACTCC GACGGCTCCT TCTTCCTCTA CAGCAAGCTC ACCGTGGACA5641 AGAGCAGGTG GCAGCAGGGG AACGTCTTCT CATGCTCCGT GATGCATGAG GCTCTGCACA5701 ACCACTACAC GCAGAAGAGC CTCTCCCTGT CTCCGGGTAA A (ii) Fc DNA sequence (mouse Igκ signal peptide underlined)(SEQ ID NO: 3, which encodes SEQ ID NO: 4) 7981                                                  ATGGA GACAGACACA8041 CTCCTGCTAT GGGTACTGCT GCTCTGGGTT CGAGGTTCCA CTGGTGACAA AACTCACACA8101 TGCCCACCGT GCCCAGCACC TGAACTCCTG GGAGGACCGT CAGTCTTCCT CTTCCCCCCA8161 AAACCCAAGG ACACCCTCAT GATCTCCCGG ACCCCTGAGG TCACATGCGT GGTGGTGGAC8221 GTGAGCCACG AAGACCCTGA GGTCAAGTTC AACTGGTACG TGGACGGCGT GGAGGTGCAT6281 AATGCCAAGA CAAAGCCGCG GGAGGAGCAG TACAACAGCA CGTACCGTGT GGTCAGCGTC8341 CTCACCGTCC TGCACCAGGA CTGGCTGAAT GGCAAGGAGT ACAAGTGCAA GGTCTCCAAC8401 AAAGCCCTCC CAGCCCCCAT CGAGAAAACC ATCTCCAAAG CCAAAGGGCA GCCCCGAGAA8461 CCACAGGTGT ACACCCTGCC CCCATCCCGC GATGAGCTGA CCAAGAACCA GGTCAGCCTG8521 ACCTGCCTGG TCAAAGGCTT CTATCCCAGC GACATCGCCG TGGAGTGGGA GAGCAATGGG8581 CAGCCGGAGA ACAACTACAA GACCACGCCT CCCGTGTTGG ACTCCGACGG CTCCTTCTTC8641 CTCTACAGCA AGCTCACCGT GGACAAGAGC AGGTGGCAGC AGGGGAACGT CTTCTCATGC8701 TCCGTGATGC ATGAGGCTCT GCACAACCAC TACACGCAGA AGAGCCTCTC CCTGTCTCCG8761 GGTAAA B. Full Length FVIIIFc(i) Full Length FVIIIFc DNA Sequence (FVIII signal peptide underlined,Fc region in bold) (SEQ ID NO: 5, which encodes SEQ ID NO: 6) 661                                         ATG CAAATAGAGC TCTCCACCTG 721 CTTCTTTCTG TGCCTTTTGC GATTCTGCTT TAGTGCCACC AGAAGATACT ACCTGGGTGC 781 AGTGGAACTG TCATGGGACT ATATGCAAAG TGATCTCGGT GAGCTGCCTG TGGACGCAAG 841 ATTTCCTCCT AGAGTGCCAA AATCTTTTCC ATTCAACACC TCAGTCGTGT ACAAAAAGAC 901 TCTGTTTGTA GAATTCACGG ATCACCTTTT CAACATCGCT AAGCCAAGGC CACCCTGGAT 961 GGGTCTGCTA GGTCCTACCA TCCAGGCTGA GGTTTATGAT ACAGTGGTCA TTACACTTAA1021 GAACATGGCT TCCCATCCTG TCAGTCTTCA TGCTGTTGGT GTATCCTACT GGAAAGCTTC1081 TGAGGGAGCT GAATATGATG ATCAGACCAG TCAAAGGGAG AAAGAAGATG ATAAAGTCTT1141 CCCTGGTGGA AGCCATACAT ATGTCTGGCA GGTCCTGAAA GAGAATGGTC CAATGGCCTC1201 TGACCCACTG TGCCTTACCT ACTCATATCT TTCTCATGTG GACCTGGTAA AAGACTTGAA1261 TTCAGGCCTC ATTGGAGCCC TACTAGTATG TAGAGAAGGG AGTCTGGCCA AGGAAAAGAC1321 ACAGACCTTG CACAAATTTA TACTACTTTT TGCTGTATTT GATGAAGGGA AAAGTTGGCA1381 CTCAGAAACA AAGAACTCCT TGATGCAGGA TAGGGATGCT GCATCTGCTC GGGCCTGGCC1441 TAAAATGCAC ACAGTCAATG GTTATGTAAA CAGGTCTCTG CCAGGTCTGA TTGGATGCCA1501 CAGGAAATCA GTCTATTGGC ATGTGATTGG AATGGGCACC ACTCCTGAAG TGCACTCAAT1561 ATTCCTCGAA GGTCACACAT TTCTTGTGAG GAACCATCGC CAGGCGTCCT TGGAAATCTC1621 GCCAATAACT TTCCTTACTG CTCAAACACT CTTGATGGAC CTTGGACAGT TTCTACTGTT1681 TTGTCATATC TCTTCCCACC AACATGATGG CATGGAAGCT TATGTCAAAG TAGACAGCTG1741 TCCAGAGGAA CCCCAACTAC GAATGAAAAA TAATGAAGAA GCGGAAGACT ATGATGATGA1801 TCTTACTGAT TCTGAAATGG ATGTGGTCAG GTTTGATGAT GACAACTCTC CTTCCTTTAT1861 CCAAATTCGC TCAGTTGCCA AGAAGCATCC TAAAACTTGG GTACATTACA TTGCTGCTGA1921 AGAGGAGGAC TGGGACTATG CTCCCTTAGT CCTCGCCCCC GATGACAGAA GTTATAAAAG1981 TCAATATTTG AACAATGGCC CTCAGCGGAT TGGTAGGAAG TACAAAAAAG TCCGATTTAT2041 GGCATACACA GATGAAACCT TTAAGACTCG TGAAGCTATT CAGCATGAAT CAGGAATCTT2101 GGGACCTTTA CTTTATGGGG AAGTTGGAGA CACACTGTTG ATTATATTTA AGAATCAAGC2161 AAGCAGACCA TATAACATCT ACCCTCACGG AATCACTGAT GTCCGTCCTT TGTATTCAAG2221 GAGATTACCA AAAGGTGTAA AACATTTGAA GGATTTTCCA ATTCTGCCAG GAGAAATATT2281 CAAATATAAA TGGACAGTGA CTGTAGAAGA TGGGCCAACT AAATCAGATC CTCGGTGCCT2341 GACCCGCTAT TACTCTAGTT TCGTTAATAT GGAGAGAGAT CTAGCTTCAG GACTCATTGG2401 CCCTCTCCTC ATCTGCTACA AAGAATCTGT AGATCAAAGA GGAAACCAGA TAATGTCAGA2461 CAAGAGGAAT GTCATCCTGT TTTCTGTATT TGATGAGAAC CGAAGCTGGT ACCTCACAGA2521 GAATATACAA CGCTTTCTCC CCAATCCAGC TGGAGTGCAG CTTGAGGATC CAGAGTTCCA2581 AGCCTCCAAC ATCATGCACA GCATCAATGG CTATGTTTTT GATAGTTTGC AGTTGTCAGT2641 TTGTTTGCAT GAGGTGGCAT ACTGGTACAT TCTAAGCATT GGAGCACAGA CTGACTTCCT2701 TTCTGTCTTC TTCTCTGGAT ATACCTTCAA ACACAAAATG GTCTATGAAG ACACACTCAC2761 CCTATTCCCA TTCTCAGGAG AAACTGTCTT CATGTCGATG GAAAACCCAG GTCTATGGAT2821 TCTGGGGTGC CACAACTCAG ACTTTCGGAA CAGAGGCATG ACCGCCTTAC TGAAGGTTTC2881 TAGTTGTGAC AAGAACACTG GTGATTATTA CGAGGACAGT TATGAAGATA TTTCAGCATA2941 CTTGCTGAGT AAAAACAATG CCATTGAACC AAGAAGCTTC TCCCAGAATT CAAGACACCC3001 TAGCACTAGG CAAAAGCAAT TTAATGCCAC CACAATTCCA GAAAATGACA TAGAGAAGAC3061 TGACCCTTGG TTTGCACACA GAACACCTAT GCCTAAAATA CAAAATGTCT CCTCTAGTGA3121 TTTGTTGATG CTCTTGCGAC AGAGTCCTAC TCCACATGGG CTATCCTTAT CTGATCTCCA3181 AGAAGCCAAA TATGAGACTT TTTCTGATGA TCCATCACCT GGAGCAATAG ACAGTAATAA3241 CAGCCTGTCT GAAATGACAC ACTTCAGGCC ACAGCTCCAT CACAGTGGGG ACATGGTATT3301 TACCCCTGAG TCAGGCCTCC AATTAAGATT AAATGAGAAA CTGGGGACAA CTGCAGCAAC3361 AGAGTTGAAG AAACTTGATT TCAAAGTTTC TAGTACATCA AATAATCTGA TTTCAACAAT3421 TCCATCAGAC AATTTGGCAG CAGGTACTGA TAATACAAGT TCCTTAGGAC CCCCAAGTAT3481 GCCAGTTCAT TATGATAGTC AATTAGATAC CACTCTATTT GGCAAAAAGT CATCTCCCCT3541 TACTGAGTCT GGTGGACCTC TGAGCTTGAG TGAAGAAAAT AATGATTCAA AGTTGTTAGA3601 ATCAGGTTTA ATGAATAGCC AAGAAAGTTC ATGGGGAAAA AATGTATCGT CAACAGAGAG3661 TGGTAGGTTA TTTAAAGGGA AAAGAGCTCA TGGACCTGCT TTGTTGACTA AAGATAATGC3721 CTTATTCAAA GTTAGCATCT CTTTGTTAAA GACAAACAAA ACTTCCAATA ATTCAGCAAC3781 TAATAGAAAG ACTCACATTG ATGGCCCATC ATTATTAATT GAGAATAGTC CATCAGTCTG3841 GCAAAATATA TTAGAAAGTG ACACTGAGTT TAAAAAAGTG ACACCTTTGA TTCATGACAG3901 AATGCTTATG GACAAAAATG CTACAGCTTT GAGGCTAAAT CATATGTCAA ATAAAACTAC3961 TTCATCAAAA AACATGGAAA TGGTCCAACA GAAAAAAGAG GGCCCCATTC CACCAGATGC4021 ACAAAATCCA GATATGTCGT TCTTTAAGAT GCTATTCTTG CCAGAATCAG CAAGGTGGAT4081 ACAAAGGACT CATGGAAAGA ACTCTCTGAA CTCTGGGGAA GGCCCCAGTC CAAAGCAATT4141 AGTATCCTTA GGACCAGAAA AATCTGTGGA AGGTCAGAAT TTCTTGTCTG AGAAAAACAA4201 AGTGGTAGTA GGAAAGGGTG AATTTACAAA GGACGTAGGA CTCAAAGAGA TGGTTTTTCC4261 AAGCAGCAGA AACCTATTTC TTACTAACTT GGATAATTTA CATGAAAATA ATACACACAA4321 TCAAGAAAAA AAAATTCAGG AAGAAATAGA AAAGAAGGAA ACATTAATCC AAGAGAATGT4381 AGTTTTGCCT CAGATACATA CAGTGACTGG CACTAAGAAT TTCATGAAGA ACCTTTTCTT4441 ACTGAGCACT AGGCAAAATG TAGAAGGTTC ATATGACGGG GCATATGCTC CAGTACTTCA4501 AGATTTTAGG TCATTAAATG ATTCAACAAA TAGAACAAAG AAACACACAG CTCATTTCTC4561 AAAAAAAGGG GAGGAAGAAA ACTTGGAAGG CTTGGGAAAT CAAACCAAGC AAATTGTAGA4621 GAAATATGCA TGCACCACAA GGATATCTCC TAATACAAGC CAGCAGAATT TTGTCACGCA4681 ACGTAGTAAG AGAGCTTTGA AACAATTCAG ACTCCCACTA GAAGAAACAG AACTTGAAAA4741 AAGGATAATT GTGGATGACA CCTCAACCCA GTGGTCCAAA AACATGAAAC ATTTGACCCC4801 GAGCACCCTC ACACAGATAG ACTACAATGA GAAGGAGAAA GGGGCCATTA CTCAGTCTCC4861 CTTATCAGAT TGCCTTACGA GGAGTCATAG CATCCCTCAA GCAAATAGAT CTCCATTACC4921 CATTGCAAAG GTATCATCAT TTCCATCTAT TAGACCTATA TATCTGACCA GGGTCCTATT4981 CCAAGACAAC TCTTCTCATC TTCCAGCAGC ATCTTATAGA AAGAAAGATT CTGGGGTCCA5041 AGAAAGCAGT CATTTCTTAC AAGGAGCCAA AAAAAATAAC CTTTCTTTAG CCATTCTAAC5101 CTTGGAGATG ACTGGTGATC AAAGAGAGGT TGGCTCCCTG GGGACAAGTG CCACAAATTC5161 AGTCACATAC AAGAAAGTTG AGAACACTGT TCTCCCGAAA CCAGACTTGC CCAAAACATC5221 TGGCAAAGTT GAATTGCTTC CAAAAGTTCA CATTTATCAG AAGGACCTAT TCCCTACGGA5281 AACTAGCAAT GGGTCTCCTG GCCATCTGGA TCTCGTGGAA GGGAGCCTTC TTCAGGGAAC5341 AGAGGGAGCG ATTAAGTGGA ATGAAGCAAA CAGACCTGGA AAAGTTCCCT TTCTGAGAGT5401 AGCAACAGAA AGCTCTGCAA AGACTCCCTC CAAGCTATTG GATCCTCTTG CTTGGGATAA5461 CCACTATGGT ACTCAGATAC CAAAAGAAGA GTGGAAATCC CAAGAGAAGT CACCAGAAAA5521 AACAGCTTTT AAGAAAAAGG ATACCATTTT GTCCCTGAAC GCTTGTGAAA GCAATCATGC5581 AATAGCAGCA ATAAATGAGG GACAAAATAA GCCCGAAATA GAAGTCACCT GGGCAAAGCA5641 AGGTAGGACT GAAAGGCTGT GCTCTCAAAA CCCACCAGTC TTGAAACGCC ATCAACGGGA5701 AATAACTCGT ACTACTCTTC AGTCAGATCA AGAGGAAATT GACTATGATG ATACCATATC5761 AGTTGAAATG AAGAAGGAAG ATTTTGACAT TTATGATGAG GATGAAAATC AGAGCCCCCG5821 CAGCTTTCAA AAGAAAACAC GACACTATTT TATTGCTGCA GTGGAGAGGC TCTGGGATTA5881 TGGGATGAGT AGCTCCCCAC ATGTTCTAAG AAACAGGGCT CAGAGTGGCA GTGTCCCTCA5941 GTTCAAGAAA GTTGTTTTCC AGGAATTTAC TGATGGCTCC TTTACTCAGC CCTTATACCG6001 TGGAGAACTA AATGAACATT TGGGACTCCT GGGGCCATAT ATAAGAGCAG AAGTTGAAGA6061 TAATATCATG GTAACTTTCA GAAATCAGGC CTCTCGTCCC TATTCCTTCT ATTCTAGCCT6121 TATTTCTTAT GAGGAAGATC AGAGGCAAGG AGCAGAACCT AGAAAAAACT TTGTCAAGCC6181 TAATGAAACC AAAACTTACT TTTGGAAAGT GCAACATCAT ATGGCACCCA CTAAAGATGA6241 GTTTGACTGC AAAGCCTGGG CTTATTTCTC TGATGTTGAC CTGGAAAAAG ATGTGCACTC6301 AGGCCTGATT GGACCCCTTC TGGTCTGCCA CACTAACACA CTGAACCCTG CTCATGGGAG6361 ACAAGTGACA GTACAGGAAT TTGCTCTGTT TTTCACCATC TTTGATGAGA CCAAAAGCTG6421 GTACTTCACT GAAAATATGG AAAGAAACTG CAGGGCTCCC TGCAATATCC AGATGGAAGA6481 TCCCACTTTT AAAGAGAATT ATCGCTTCCA TGCAATCAAT GGCTACATAA TGGATACACT6541 ACCTGGCTTA GTAATGGCTC AGGATCAAAG GATTCGATGG TATCTGCTCA GCATGGGCAG6601 CAATGAAAAC ATCCATTCTA TTCATTTCAG TGGACATGTG TTCACTGTAC GAAAAAAAGA6661 GGAGTATAAA ATGGCACTGT ACAATCTCTA TCCAGGTGTT TTTGAGACAG TGGAAATGTT6721 ACCATCCAAA GCTGGAATTT GGCGGGTGGA ATGCCTTATT GGCGAGCATC TACATGCTGG6781 GATGAGCACA CTTTTTCTGG TGTACAGCAA TAAGTGTCAG ACTCCCCTGG GAATGGCTTC6841 TGGACACATT AGAGATTTTC AGATTACAGC TTCAGGACAA TATGGACAGT GGGCCCCAAA6901 GCTGGCCAGA CTTCATTATT CCGGATCAAT CAATGCCTGG AGCACCAAGG AGCCCTTTTC6961 TTGGATCAAG GTGGATCTGT TGGCACCAAT GATTATTCAC GGCATCAAGA CCCAGGGTGC7021 CCGTCAGAAG TTCTCCAGCC TCTACATCTC TCAGTTTATC ATCATGTATA GTCTTGATGG7081 GAAGAAGTGG CAGACTTATC GAGGAAATTC CACTGGAACC TTAATGGTCT TCTTTGGCAA7141 TGTGGATTCA TCTGGGATAA AACACAATAT TTTTAACCCT CCAATTATTG CTCGATACAT7201 CCGTTTGCAC CCAACTCATT ATAGCATTCG CAGCACTCTT CGCATGGAGT TGATGGGCTG7261 TGATTTAAAT AGTTGCAGCA TGCCATTGGG AATGGAGAGT AAAGCAATAT CAGATGCACA7321 GATTACTGCT TCATCCTACT TTACCAATAT GTTTGCCACC TGGTCTCCTT CAAAAGCTCG7381 ACTTCACCTC CAAGGGAGGA GTAATGCCTG GAGACCTCAG GTGAATAATC CAAAAGAGTG7441 GCTGCAAGTG GACTTCCAGA AGACAATGAA AGTCACAGGA GTAACTACTC AGGGAGTAAA7501 ATCTCTGCTT ACCAGCATGT ATGTGAAGGA GTTCCTCATC TCCAGCAGTC AAGATGGCCA7561 TCAGTGGACT CTCTTTTTTC AGAATGGCAA AGTAAAGGTT TTTCAGGGAA ATCAAGACTC7621 CTTCACACCT GTGGTGAACT CTCTAGACCC ACCGTTACTG ACTCGCTACC TTCGAATTCA7681 CCCCCAGAGT TGGGTGCACC AGATTGCCCT GAGGATGGAG GTTCTGGGCT GCGAGGCACA7741 GGACCTCTAC GACAAAACTC ACACATGCCC ACCGTGCCCA GCTCCAGAAC TCCTGGGCGG7801 ACCGTCAGTC TTCCTCTTCC CCCCAAAACC CAAGGACACC CTCATGATCT CCCGGACCCC7861 TGAGGTCACA TGCGTGGTGG TGGACGTGAG CCACGAAGAC CCTGAGGTCA AGTTCAACTG7921 GTACGTGGAC 0GCGTGGAGG TGCATAATGC CAAGACAAAG CCGCGGGAGG AGCAGTACAA7981 CAGCACGTAC CGTGTGGTCA GCGTCCTCAC CGTCCTGCAC CAGGACTGGC TGAATGGCAA8041 GGAGTACAAG TGCAAGGTCT CCAACAAAGC CCTCCCAGCC CCCATCGAGA AAACCATCTC8101 CAAAGCCAAA GGGCAGCCCC GAGAACCACA GGTGTACACC CTGCCCCCAT CCCGGGATGA8161 GCTGACCAAG AACCAGGTCA GCCTGACCTG CCTGGTCAAA GGCTTCTATC CCAGCGACAT8221 CGCCGTGGAG TGGGAGAGCA ATGGGCAGCC GGAGAACAAC TACAAGACCA CGCCTCCCGT8281 GTTGGACTCC GACGGCTCCT TCTTCCTCTA CAGCAAGCTC ACCGTGGACA AGAGCAGGTG8341 GCAGCAGGGG AACGTCTTCT CATGCTCCGT GATGCATGAG GCTCTGCACA ACCACTACAC8401 GCAGAAGAGC CTCTCCCTGT CTCCGGGTAA A(ii) Fc (same sequence as A (ii) (SEQ ID NO: 3))]

TABLE 11 Polypeptide SequencesA. B-Domain Deleted FVIII-Fc Monomer Hybrid (BDD FVIIIFcmonomer dimer): created by coexpressing BDD FVIIIFc and Fcchains. Construct = HC-LC-Fc fusion. An Fc expression cassetteis cotransfected with BDDFVIII-Fc to generate the BDD FVIIIFcmonomer-. For the BDD FVIIIFc chain, the Fc sequence is shownin bold; HC sequence is shown in double underline; remaining Bdomain sequence is shown in italics. Signal peptides are underlined.i) B domain deleted FVIII-Fc chain (19 amino acid signalsequence underlined) (SEQ ID NO: 2) MQIELSTCFFLCLLRFCFSATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPR SFSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLPGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQPSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQPLYDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVQGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKii) Fc chain (20 amino acid heterologous signal peptide frommouse Igκ chain underlined) (SEQ ID NO: 4) METDTLLLWVLLLWVPGSTGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALDAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKB. Full length FVIIIFc monomer hybrid (Full length FVIIIFcmonomer dimer): created by coexpressing FVIIIFc and Fc chains.Construct = HC-B-LC-Fc fusion. An Fc expression cassette iscotransfected with full length FVIII-Fc to generate the fulllength FVIIIFc monomer. For the FVIIIFc chain, the Fc sequenceis shown in bold; HC sequence is shown in double underline;B domain sequence is shown in italics. Signal peptides are underlined.i) Full length FVIIIFc chain (FVIII signal peptide underlined(SEQ ID NO: 6) MQIELSTCFFECLERFCFSATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIER SFSQNSRHPSTRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSDLLMLLRQSPTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQLRLNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTTLFGKKSSPLTESGGPLSLSEENNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHGPALLTKDNALFKVSISLLKTNKTSNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTPLIHDRMLMDKNATALRLNHMSNKTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKMLFLPESARWIQRTHGKNSLNSGQGPSPKQLVSLGPEKSVEGQNFLSEKNKVVVGKGEFTKDVGLKEMVFPSSRNLFLTNLDNLHENNTHNQEKKIQEEIEKKETLIQENVVLPQIHTVTGTKNFMKNLELLSTRQNVEGSYDGAYAPVLQDFRSLNDSTNRTKKHTAHFSKKGEEENLEGLGNQTKQIVEKYACTTRISPNTSQQNFVTQRSKRALKQFRLPLEETELEKRHVDDTSTQWSKNMKHLTPSTLTQIDYNEKEKGAITQSPLSDCLTRSHSIPQANRSPLPIAKVSSFPSIRPIYLTRVLFQDNSSHLPAASYRKKDSGVQESSHFLQGAKKNNLSLAILTLEMTGDQREVGSLGTSATNSVTYKKVENTVLPKPDLPKTSGKVELLPKVHIYQKDLFPTETSNGSPGHLDLVEGSLLQGTEGAIKWNEANRPGKVPFLRVATESSAKTPSKLLDPLAWDNHYGTQIPKEEWKSQEKSPEKTAFKKKDTILSLNACESNHAIAAINEGQNKPEIEVTWAKQGRTERLCSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHFGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYIGQWAPKLARLHYSGSINAWSTKEPFSWIKYDLLAPMIIHGKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRVVQQGNVFSCSVMHEALUNHYTQKSLSLSPGKii) Fc chain (20 amino acid heterologous signal peptidefrom mouse Igκ chain underlined) (SEQ ID NO: 4) METDTLLLWVLLLWVPGSTGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingknowledge within the skill of the art, readily modify and/or adapt forvarious applications such specific embodiments, without undueexperimentation, without departing from the general concept of thepresent invention. Therefore, such adaptations and modifications areintended to be within the meaning and range of equivalents of thedisclosed embodiments, based on the teaching and guidance presentedherein. It is to be understood that the phraseology or terminologyherein is for the purpose of description and not of limitation, suchthat the terminology or phraseology of the present specification is tobe interpreted by the skilled artisan in light of the teachings andguidance.

Other embodiments of the invention will be apparent to those skilled inthe art from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

All patents and publications cited herein are incorporated by referenceherein in their entirety.

1. A method of reducing the annualized bleeding rate in a subject havinghemophilia comprising administering to the subject an effective dose ofa long-acting FVIII polypeptide at a dosing interval of about three daysor longer. 2-7. (canceled)
 8. The method of claim 1, wherein theeffective dose is between about 20 IU/kg to about 90 IU/kg. 9-44.(canceled)
 45. A method of controlling or preventing bleeding orbleeding episodes in a subject in need of surgical prophylaxis,peri-operative management, or treatment for surgery, the methodcomprising administering to the subject a pharmaceutical compositioncomprising an effective dose of a long-acting FVIII polypeptide; whereinthe pharmaceutical composition is provided as a lyophilized powdersuitable for administration following reconstitution with sterile waterfor injection, wherein the lyophilized powder upon reconstitution withabout 3 mL sterile water for injection (WFI) produces a solutioncomprising: (a) about 50 IU/mL to about 2500 IU/mL of a long-actingFVIII polypeptide; (b) about 13 mg/mL to about 20 mg/mL sucrose; (c)about 10 mg/mL to about 13 mg/mL sodium chloride (NaCl); (d) about 0.75mg/mL to about 2.25 mg/mL L-histidine; (e) about 5 mM to about 10 mMcalcium chloride; and (f) about 0.08 mg/mL to about 0.25 mg/mLpolysorbate 20 or polysorbate 80, and wherein the long-acting FVIIIpolypeptide comprises a first subunit and a second subunit, wherein thefirst subunit comprises a FVIII protein and a first FcRn bindingpartner, and wherein the second subunit comprises a second FcRn bindingpartner.
 46. (canceled)
 47. The method of claim 45, wherein the subjectis in need of management of bleeding associated with surgery or dentalextraction.
 48. The method of claim 45, wherein the subject willundergo, is undergoing, or has undergone major surgery.
 49. The methodof claim 45, wherein the major surgery is orthopedic surgery, extensiveoral surgery, urologic surgery, or hernia surgery.
 50. The method ofclaim 49, wherein the orthopedic surgery is replacement of knee, hip, orother major joint. 51-116. (canceled)
 117. The method of claim 45,wherein the effective dose is between 25 IU/kg to about 60 IU/kg. 118.The method of claim 45, wherein the effective dose is 25 IU/kg.
 119. Themethod of claim 45, wherein the effective dose is 30 IU/kg.
 120. Themethod of claim 45, wherein the effective dose is 35 IU/kg.
 121. Themethod of claim 45, wherein the effective dose is 40 IU/kg.
 122. Themethod of claim 45, wherein the effective dose is 45 IU/kg.
 123. Themethod of claim 45, wherein the effective dose is 50 IU/kg.
 124. Themethod of claim 45, wherein the effective dose is 55 IU/kg.
 125. Themethod of claim 45, wherein the effective dose is 60 IU/kg.
 126. Themethod of claim 45, wherein the effective dose is between 25 IU/kg to 40IU/kg.
 127. The method of claim 45, wherein the effective dose isbetween 40 IU/kg to 60 IU/kg.
 128. The method of claim 45, wherein theeffective dose is between 25 IU/kg to about 60 IU/kg; and wherein theeffective dose is administered at a dosing interval of about every 12 to24 hours.
 129. A pharmaceutical composition comprising: a. a long-actingFVIII polypeptide comprising one or more heterologous FcRn bindingdomain; b. one or more stabilizing agents selected from the groupconsisting of sucrose, trehalose, raffinose, arginine, and anycombination thereof, c. sodium chloride (NaCl); d. L-histidine; e.calcium chloride; and f. polysorbate 20 or polysorbate 80.